Patent application title: Enzymatic synthesis of deoxyribonucleosides
Inventors:
Wilhelm Tischer (Peibenberg, DE)
Hans-Georg Ihlenfeldt (Iffeldorf, DE)
Octavian Barzu (Antony, FR)
Hiroshi Sakamoto (Meudon, FR)
Elisabeth Pistotnik (Creteil, FR)
Philippe Marliere (Etiolles, FR)
Sylvie Pochet (Paris, FR)
IPC8 Class: AC12P1934FI
USPC Class:
435 911
Class name: N-glycoside nucleotide polynucleotide (e.g., nucleic acid, oligonucleotide, etc.)
Publication date: 2008-11-13
Patent application number: 20080280329
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Patent application title: Enzymatic synthesis of deoxyribonucleosides
Inventors:
Philippe Marliere
Wilhelm Tischer
Hans-Georg Ihlenfeldt
Octavian Barzu
Hiroshi Sakamoto
Elisabeth Pistotnik
Sylvie Pochet
Agents:
FULBRIGHT & JAWORSKI, LLP
Assignees:
Origin: NEW YORK, NY US
IPC8 Class: AC12P1934FI
USPC Class:
435 911
Abstract:
The present invention relates to a method for the in vitro synthesis of
deoxyribonucleosides and enzymes suitable for this method.Claims:
1-45. (canceled)
46. A method for in vitro enzymatic synthesis of a deoxyribonucleoside comprising reacting deoxyribose 1-phosphate (dR1P) and a nucleobase to form a deoxyribonucleoside and an inorganic phosphate.
47. The method of claim 46, further comprising removing the inorganic phosphate.
48. The method of claim 46, wherein said reacting comprises catalyzing said dR1P and said nucleobase with a thymidine phosphorylase (TP, EC 2.4.2.4.) or a purine nucleoside phosphorylase (PNP, EC 2.4.2.1.).
49. The method of claim 47, comprising removing the inorganic phosphate by one of: (i) converting the inorganic phosphate to inorganic pyrophosphate, (ii) precipitating the inorganic phosphate, (iii) complexing the inorganic phosphate, or (iv) phosphorylating a substrate with the inorganic phosphate.
50. The method of claim 47, comprising removing the inorganic phosphate by phosphorylating a substrate with the inorganic phosphate.
51. The method of claim 46, wherein the nucleobase is selected from the group consisting of thymine, uracil, adenine, guanine, hypoxanthinine and an analog thereof.
52. The method of claim 51, wherein said analog is selected from the group consisting of: 2-thio-uracil, 6-aza-uracil, 5-carboxy-2-thio-uracil, 6-aza-thymine, 6-aza-2-thio-thymine and 2,6-diamino-purine.
53. The method of claim 46, further comprising reacting said inorganic phosphate with fructose-diphosphate (FDP) to form pyrophosphate and fructose-6-phosphate (F6P).
54. The method of claim 53, wherein said reacting comprises catalyzing said inorganic phosphate with said FDP with a Ppi-dependent phosphofructokinase (PFK-Ppi, EC 2.7.1.90).
55. The method of claim 46, further comprising reacting said inorganic phosphate with a disaccharide to form a monosaccharide and a phosphorylated monosaccharide.
56. The method of claim 55, wherein the disaccharide is sucrose or maltose.
57. The method of claim 56, wherein said reacting comprises catalyzing said inorganic phosphate and said disaccharide with a sucrose phosphorylase (EC 2.4.1.7) or a maltose phosphorylase (EC 2.4.1.8).
58. The method of claim 46, further comprising generating dR1P by isomerizing deoxyribose 5-phosphate (dR5P) prior to reacting said dR1P with a nucleobase.
59. The method of claim 58, comprising isomerizing said dR5P with a phosphopentose mutase (PPM, EC 5.4.2.7).
60. The method of claim 58, further comprising forming the dR5P by condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde prior to isomerizing said dR5P.
61. The method of claim 60, wherein said condensing comprises catalyzing condensing of GAP with acetaldehyde with a phosphopentose aldolase (PPA, EC 4.1.2.4).
62. The method of claim 60, further comprising enzymatically generating said GAP from fructose 1,6-diphosphate, dihydroxyacetone (DHA) or glycerolphosphate (GP) prior to said condensing.
63. The method of claim 58, comprising generating said deoxyribose 5-phosphate by phosphorylating deoxyribose prior to isomerizing said dR5P.
64. The method of claim 63, wherein said phosphorylating comprises catalyzing of deoxyribose with a deoxyribokinase (dRK, EC 2.7.1.15.).
65. The method of claim 64, wherein said dRK is encoded by (a) the nucleotide sequence of SEQ ID NO: 11, (b) a nucleotide sequence encoding the protein encoded by SEQ ID NO: 11 or (c) a nucleotide sequence which hybridizes under stringent conditions to the complementary sequence of (a) or (b).
66. The method of claim 62, comprising reacting fructose 1,6-diphosphate with an FDP-aldolase I or an FDP-aldolase II to form said GAP.
67. The method of claim 62, comprising forming said GAP by reacting DHA with ATP to form dihydroxyacetone phosphate (DHAP), followed by catalyzing isomerization of said DHAP to GAP with a glycerokinase (GK, EC 2.7.1.30) and a triose phosphate isomerase (TIM, EC 5.3.1.1).
68. The method of claim 62, comprising forming said GAP by reacting GP with O2 to form dihydroxyacetone phosphate (DHAP) and H2O2, followed by catalyzing isomerization of said DHAP to GAP with a glycerophosphate oxidase (GPO, EC 1.1.3.21) and a triose phosphate isomerase (TIM, EC 5.3.1.1).
69. The method of claim 46, further comprising reacting said deoxyribonucleoside with a second nucleobase to form a second deoxyribonucleoside containing the second nucleobase.
70. The method of claim 69, comprising catalyzing said reacting with a nucleoside 2-deoxyribosyl transferase (NdT, EC 2.4.2.6).
71. The method of claim 70, wherein said NdT is encoded by (a) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 13, (b) a nucleic acid molecule consisting of a nucleotide sequence encoding the protein encoded by SEQ ID NO: 13 or (c) a nucleic acid molecule which hybridizes under stringent conditions to the nucleic acid molecule of (a) or (b).
72. The method of claim 69, wherein said second nucleobase is selected from the group consisting of cytosine and a cytosine analog.
73. The method of claim 69, wherein said second nucleobase is selected from the group consisting of 5-aza-cytosine, 2,6-dichloro-purine, 6-aza-thymine and 5-fluoro-uracil.
74. A method for the in vitro enzymatic synthesis of a deoxyribonucleoside comprising:(i) condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde to form deoxyribose 5-phosphate (dR5P),(ii) isomerizing said dR5P to deoxyribose 1-phosphate (dR1P), and(iii) reacting said dR1P and a nucleobase, to form said deoxyribonucleoside and an inorganic phosphate.
75. The method of claim 74, further comprising removing the inorganic phosphate.
76. The method of claim 75, comprising removing the inorganic phosphate by phosphorylation of a substrate with the inorganic phosphate.
77. The method of claim 74, comprising carrying out the complete reaction of steps (i) to (iii) without isolating intermediate products.
78. The method of claim 74, comprising generating said GAP from fructose 1,6-diphosphate (FDP), dihydroxy-acetone (DHA) or glycerolphosphate (GP) prior to said condensing of GAP.
79. The method of claim 74, further comprising removing excess acetaldehyde before step (ii).
80. The method of claim 78, further comprising generating said GAP and removing excess starting materials or by-products before step (ii).
81. The method of claim 80, wherein said excess starting material is fructose 1,6-diphosphate and said excess by-product is deoxyxylulose 1-phosphate (dX1P).
82. The method of claim 78, comprising generating GAP from FDP, and generating DXP1 as an excess by-product thereby.
83. A method for the in vitro enzymatic synthesis of a deoxyribonucleoside comprising:(i) phosphorylating deoxyribose to deoxyribose 5-phosphate (dR5P),(ii) isomerizing said dR5P to deoxyribose 1-phosphate (dR1P), and(iii) reacting said dR1P and a nucleobase to form said deoxyribonucleoside and an inorganic phosphate.
84. The method of claim 83, further comprising removing the inorganic phosphate.
85. The method of claim 84, comprising removing the inorganic phosphate by phosphorylating a substrate with the inorganic phosphate.
86. The method of claim 83, comprising conducting the complete reaction of steps (i) to (iii) without isolating intermediate products.
87. A method for preparing an enzyme for an in vitro method for enzymatic synthesis of a deoxyribonucleoside, comprising reacting (i) an isolated nucleic acid molecule encoding a nucleoside 2-deoxyribosyl transferase (NdT, EC 2.4.2.6) with (ii) a deoxyribonucleoside containing a first nucleobase, wherein said nucleic acid molecule comprises (a) the nucleotide sequence shown in SEQ ID NO: 13, (b) a nucleotide sequence encoding the protein encoded by SEQ ID NO: 13 or (c) a nucleotide sequence hybridizing under stringent conditions to the complementary sequence of (a) or (b), and wherein said deoxyribonucleoside containing a first nucleobase is further reacted with a second nucleobase to form a deoxyribonucleoside containing said second nucleobase.
88. The method of claim 87, wherein the second nucleobase is selected from the group consisting of cytidine and a cytidine analog.
89. The method of claim 88, wherein the analog is selected from the group consisting of: 6-methyl purine, 2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 5-azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-azathymine, 5-fluorouracil, ethyl-4-amino-5-imidazole carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide.
90. The method of claim 87, wherein the first nucleobase is selected from the group consisting of adenine, guanine, thymine, uracil and hypoxanthine.
91. The method of claim 87, comprising containing the nucleic acid molecule in a recombinant vector in operative linkage with an expression control sequence.
92. The method of claim 81, comprising containing the nucleic acid in a recombinant cell.
93. A method for preparing an enzyme for an in vitro method for enzymatic synthesis of a deoxyribonucleoside, comprising reacting (i) an isolated nucleic acid molecule encoding a deoxyribokinase (dRK, EC 2.7.1.5) with (ii) deoxyribose, further comprising phosphorylating said deoxyribose to deoxyribose 5-phosphate, wherein said nucleic acid molecule comprises (a) the nucleotide sequence shown in SEQ ID NO: 11, (b) a nucleotide sequence encoding the protein encoded by SEQ ID NO: 11 or (c) a nucleotide sequence hybridizing under stringent conditions to the complementary sequence of (a) or (b).
94. A method for synthesizing a deoxyribonucleoside in vitro, comprising contacting a mixture containing deoxyribose and phosphate with an enzyme having NdT activity to form deoxyribose 5-phosphate and obtaining deoxyribose 5-phosphate therefrom.
Description:
RELATED APPLICATIONS
[0001]This application is a divisional application of U.S. Ser. No. 10/049,750 filed Dec. 9, 2002, incorporated herein by reference in its entirety, which is a §371 of PCT/EP00/08088 filed Aug. 18, 2000, which claims priority from European Patent Application No. 99 116 425.2 filed Aug. 20, 1999.
DESCRIPTION
[0002]The present invention relates to a method for the in vitro enzymatic synthesis of deoxyribonucleosides and enzymes suitable for this method.
[0003]Natural deoxyribonucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC and thymidine, dT) are building blocks of DNA. The N-glycosidic bond between nucleobase and sugar involves the N1 of a pyrimidine or the N9 of a purine ring and the C1, of deoxyribose.
[0004]In the living cells the four deoxyribonucleosides (dN) result from the "salvage pathway" of nucleotide metabolism. A group of enzymes is involved in cellular catabolism of deoxyribonucleosides. Besides deoxyriboaldolase (EC 4.1.2.4) and deoxyribomutase (EC 5.4.2.7), this group also includes thymidine phosphorylase (EC 2.4.2.4) and purine nucleoside phosphorylase (EC 2.4.2.1). These four enzymes are induced by the addition of deoxyribonucleosides to the growth medium. The genes coding for these enzymes have been shown to map closely together on the bacterial chromosome (Hammer-Jesperson and Munch-Peterson, Eur. J. Biochem. 1 7 (1970), 397 and literature cited therein). In E. coli the genes as described above are located on the deo operon which exhibits an unusual and complicated pattern of regulation (Valentin-Hansen et al., EMBO J. 1 (1982), 317).
[0005]Using the enzymes of the deo operon for synthesis of deoxynucleosides was described by C. F. Barbas III (Overproduction and Utilization of Enzymes in Synthetic Organic Chemistry, Ph.D. Thesis (1989), Texas A&M University). He applied phosphopentomutase and thymidine phosphorylase for the synthesis of deoxynucleosides. Deoxyribose 5-phosphate was prepared by chemical synthesis (Barbas III et al., J. Am. Chem. Soc. 112 (1990), 2013-2014), which makes this compound expensive as starting material and not suitable for large scale synthesis. He also made deoxyriboaldolase available as a recombinant enzyme and investigated its synthetic applicability but neither he nor C. H. Wong (Microbial Aldolases in Carbohydrate Synthesis: ACS Symp. Ser. No. 466: Enzymes in Carbohydrate Synthesis, Eds. M. D. Bednarski, E. S. Simon (1991), 23-27) were able to carry out a coupled one-pot synthesis employing all three enzymes. It appears likely that some drawbacks exist which could not be circumvented. Among these drawbacks are insufficient chemical equilibrium, instability of intermediates, such as deoxyribose 1-phosphate and inactivation and inhibition effects of involved compounds on the enzymes.
[0006]Evidence of an advantageous equilibrium is given by S. Roy et al. (JACS 108 (1986), 1675-78). For the aldolase reaction the equilibrium is on the desired product side (deoxyribose 5-phosphate), for the phosphopentomutase it is on the wrong side (also deoxyribose 5-phosphate) and for the purine nucleoside phosphorylase it is on the desired synthesis product side. The authors suggest coupling of the three enzyme reactions to obtain reasonable yields. Contrary to these suggestions they prepared deuterated deoxyguanosine and thymidine in a two step procedure, that is deoxyribose 5-phosphate in a first step and deoxynucleoside in a second step. Isolated yields of the second step were 11% and 5% for deoxyguanosine and thymidine, respectively. These low yields are also obtained in the preparation of arabinose-based nucleosides (Barbas III (1990), supra). These low yields indicate serious drawbacks for the use of the enzymes of the deo operon in a synthetic route which have to work in the reverse direction of their biological function, which is degradation of deoxynucleosides.
[0007]Thus, there does not exist any economical commercial method at present for the enzymatic in vitro synthesis of deoxyribonucleosides. Hitherto, for commercial purposes, deoxynucleosides are generated from fish sperm by enzymatic cleavage of DNA. This method, however, involves several disadvantages, particularly regarding difficulties of obtaining the starting material in sufficient quantity and quality.
[0008]Therefore, it was an object of the invention to provide a method, by means of which the drawbacks of the prior are eliminated at least partially and which allows efficient and economical synthesis of deoxyribonucleosides without any dependence on unreliable natural sources.
[0009]Surprisingly, it was found that the drawbacks of previous enzymatic synthesis routes can be avoided and deoxyribonucleosides can be obtained in high yields of e.g. at least 80% based on the amount of starting material.
[0010]In a first aspect, the present invention relates to a method for the in vitro enzymatic synthesis of deoxyribonucleosides comprising reacting deoxyribose 1-phosphate (dR1P) and a nucleobase, wherein a deoxyribonucleoside and inorganic phosphate are formed.
[0011]The reaction is catalyzed by an enzyme which is capable of transferring a deoxyribose moiety to a nucleobase, with a deoxyribonucleoside being formed. Preferably, the reaction is catalyzed by a thymidine phosphorylase (TP, EC 2.4.2.4) or a purine nucleoside phosphorylase (PNP, EC 2.4.2.1). For the EC designation of these enzymes and other enzymes mentioned below reference is made to the standard volume Enzyme Nomenclature 1992, Ed. E. C. Webb, Academic Press, Inc.
[0012]These enzymes and other enzymes mentioned below are obtainable as native proteins from natural sources, i.e. any suitable organisms selected from eukaryotes, prokaryotes and archaea including thermophilic organisms. Further, these enzymes are obtainable as recombinant proteins from any suitable host cell which is transformed or transfected with a DNA encoding said enzyme. The host cell may be a eukaryotic cell, a prokaryotic cell or an archaea cell. Particular preferred sources of native or recombinant TP or PNP are prokaryotic organisms such as E. coli. Recombinant TP may be isolated from E. coli strain pHSP 282 (CNCM I-21 86) deposited on Apr. 23, 1999, which is a recombinant E. coli strain transformed with a plasmid containing the E. coli deoA (thymidine phosphorylase) insert. Recombinant PNP may be isolated from E. coli strain pHSP 283 (CNCM 1-2187) deposited on Apr. 23, 1999, which is a recombinant E. coli strain transformed with a plasmid containing the E. coli deoD (purine nucleoside phosphorylase) insert. The nucleotide sequence of the TP gene and the corresponding amino acid sequence are shown in SEQ ID NO.1 and 2. The nucleotide sequence of the PNP gene and the corresponding amino acid sequence are shown in SEQ ID NO.15 and 16 and 3 and 4.
[0013]The nucleobase, to which the deoxyribose unit is transferred, will be selected from any suitable nucleobase. For example, the nucleobase may be a naturally occurring nucleobase such as thymine, uracil, adenine, guanine or hypoxanthine. It should be noted, however, that also non-naturally occurring analogs thereof are suitable as enzyme substrates such as 2-thio-uracil, 6-aza-uracil, 5-carboxy-2-thiouracil, 6-aza-thymine, 6-aza-2-thio-thymine and 2,6-diamino-purine.
[0014]Preferably the inorganic phosphate is removed from the reaction. This removal is preferably effected by (i) conversion to inorganic pyrophosphate, (ii) precipitation/complexation and/or (iii) substrate phosphorylation.
[0015]Conversion to inorganic pyrophosphate may be effected by a phosphate transfer from a phosphorylated, preferably polyphosphorylated substrate such as fructose diphosphate (FDP), wherein a phosphate group is cleaved from the phosphorylated substrate and reacts with the inorganic phosphate, with inorganic pyrophosphate (PPi) being formed. This phosphate transfer is preferably catalyzed by a PPi-dependent phosphorylase/kinase, e.g. by a PPi-dependent phosphofructokinase (PFK-PPi, EC 2.7.1.90), which catalyzes the reaction of fructose diphosphate (FDP) and inorganic phosphate to fructose 6-phosphate (F6P) and inorganic pyrophosphate. Preferred sources of PPi-dependent kinases/phosphorylases and genes coding therefor are from Propionibacterium freudenreichii (shermanii) or from potato tubers.
[0016]Further, the inorganic phosphate may be removed from the reaction by precipitation and/or complexation which may be effected by adding polyvalent metal ions, such as calcium or ferric ions capable of precipitating phosphate or by adding a complex-forming compound capable of complexing phosphate. It should be noted that also a combination of pyrophosphate formation and complexation/precipitation may be carried out.
[0017]Furthermore, the removal of inorganic phosphate may be effected by substrate phosphorylation. Thereby the inorganic phosphate is transferred to a suitable substrate, with a phosphorylated substrate being formed. The substrate is preferably selected from saccharides, e.g. disaccharides such as sucrose or maltose. When using disaccharides as substrate, a monosaccharide and a phosphorylated monosaccharide are obtained. The phosphate transfer is catalyzed by a suitable phosphorylase/kinase such as sucrose phosphorylase (EC 2.4.1.7) or maltose phosphorylase (EC 2.4.1.8). Preferred sources of these enzymes are Leuconostoc mesenteroides, Pseudomonas saccherophila (sucrose phosphorylase) and Lactobacillus brevis (maltose phosphorylase).
[0018]The phosphorylated substrate may be further reacted by additional coupled enzymatic reactions, e.g. into a galactoside (Ichikawa et al., Tetrahedron Lett. 36 (1995), 8731-8732). Further, it should be noted that phosphate removal by substrate phosphorylation may also be coupled with other phosphate removal methods as described above.
[0019]Deoxyribose 1-phosphate (dR1P), the starting compound of the method of the invention, is a rather unstable compound, the isolation of which is difficult. In a preferred embodiment of the present invention, d1RP is generated in situ from deoxyribose 5-phosphate (dR5P) which is relatively stable at room temperature and neutral pH. This reaction is catalyzed by a suitable enzyme, e.g. a deoxyribomutase (EC 5.4.2.7) or a phosphopentose mutase (PPM, EC 5.4.2.7) which may be obtained from any suitable source as outlined above. The reaction is preferably carried out in the presence of divalent metal cations, e.g. Mn2+ or Co2+ as activators. Preferred sources of deoxyribomutase are enterobacteria. Particular preferred sources of native or recombinant PPM are prokaryotic organisms such as E. coli. Recombinant PPM may be isolated from E. coli strain pHSP 275 (CNCM I-2188) deposited on Apr. 23, 1999, which is a recombinant E. coli strain transformed with a plasmid containing the E. coli deo B (phosphopentose mutase) insert. The nucleotide sequence of the PPM gene and the corresponding amino acid sequence are shown in SEQ ID NO.17 and 18 and 5 and 6.
[0020]dR5P may be generated by a condensation of glyceraldehyde 3-phosphate (GAP) with acetaldehyde. This reaction is catalyzed by a suitable enzyme, preferably by a phosphopentose aldolase (PPA, EC 4.1.2.4). The reaction exhibits an equilibrium constant favorable to the formation of the phosphorylated sugar (Keq=[dR5P]/[acetaldehyde]×[GAP]=4.2×103×M.su- p.-1). PPA forms an unstable Schiff base intermediate by interacting with the aldehyde. Particular preferred sources of native or recombinant PPA are prokaryotic organisms such as E. coli. Recombinant PPA may be isolated from E. coli strain pHSP 276 (CNCM I-2189) deposited on Apr. 23, 1999. This recombinant E. coli strain is transformed with a plasmid containing the deoC (phosphopentosealdolase) insert. The nucleotide sequence of the PPA gene and the corresponding amino acid sequence are shown in SEQ ID NO.19 and 20 and 7 and 8.
[0021]GAP is a highly unstable compound and, thus, should be generated in situ from suitable precursors which are preferably selected from fructose 1,6-diphosphate (FDP), dihydroxyacetone (DHA) and/or glycerolphosphate (GP), with FDP being preferred.
[0022]FDP can be converted by an FDP aldolase (EC 4.1.2.13) selected from FDP aldolases I and FDP aldolases II to GAP and dihydroxyacetone phosphate (Keq=[FDP]/[GAP]×[DHAP]=104M-1). The two families of FDP aldolases giving identical end products (GAP and DHAP) via two chemically distinct pathways may be used for this reaction. FDP aldolase I forms Schiff base intermediates like PPA, and FDP aldolase II which uses metals (Zn2+) covalently bound to the active sites to generate the end products. FDP-aldolase I is characteristic to eukaryotes, although it is found in various bacteria. FDP-aldolase II is more frequently encountered in prokaryotic organisms. If FDP-aldolase reacts with FDP in the presence of acetaldehyde, the latter compound can interact with DHAP to yield an undesired condensation by-product named deoxyxylolose 1-phosphate (dX1P). Thus, the reaction is preferably conducted in a manner by which the generation of undesired side products is reduced or completely suppressed.
[0023]Particular preferred sources of native or recombinant FDP aldolases are prokaryotic or eukaryotic organisms. For example, FDP aldolase may be isolated from rabbit muscle. Further, FDP aldolase may be obtained from bacteria such as E. coli. Recombinant FDP aldolase may be isolated from recombinant E. coli strain pHSP 284 (CNCM I-2190) which is transformed with a plasmid containing the E. coli fba (fructose diphosphate aldolase) insert. The nucleotide sequence of the E. coli FDP aldolase gene and the corresponding amino acid sequence are shown in SEQ ID NO.9 and 10.
[0024]On the other hand, GAP may be generated from DHAP and ATP, with dihydroxyacetone phosphate (DHAP) and ADP being formed and subsequent isomerization of DHAP to GAP in a reaction catalyzed by a glycerokinase (GK, EC 2.7.1.30) and a triose phosphate isomerase (TIM, EC 5.3.1.1). Suitable glycerokinases are obtainable from E. coli, suitable triose phosphate isomerases are obtainable from bovine or porcine muscle.
[0025]In a still further embodiment of the present invention GAP may be generated from glycerol phosphate (GP) and O2, with DHAP and H2O2 being formed and subsequent isomerization of DHAP to GAP in a reaction catalyzed by a glycerophosphate oxidase (GPO, EC 1.1.3.21) and a triose phosphate isomerase (TIM, EC 5.3.1.1). Suitable glycerophosphate oxidases are obtainable from Aerococcus viridans.
[0026]In an alternative embodiment of the present invention deoxyribose 5-phosphate (dR5P) is generated by phosphorylation of deoxyribose. Preferably this reaction is carried out in the presence of a suitable enzyme, e.g. a deoxyribokinase (dRK, EC 2.7.1.5) which may be obtained from prokaryotic organisms, particularly Salmonella typhi and in the presence of ATP. The nucleotide sequence of the Salmonella dRK gene and the corresponding amino acid sequence are shown in SEQ ID NO.11 and 12.
[0027]By the reaction as outlined above deoxyribonucleosides are obtained which contain a nucleobase which is accepted by the enzymes TP and/or PNP. TP is specific for thymidine (T), uracil (U) and other related pyrimidine compounds. PNP uses adenine, guanine, hypoxanthine or other purine analogs as substrates.
[0028]The synthesis of deoxyribonucleosides which are not obtainable by direct condensation such as deoxycytosine (dC), thus, require an additional enzymatic reaction, wherein a deoxyribonucleoside containing a first nucleobase is reacted with a second nucleobase, with a second ribonucleoside containing the second nucleobase being formed. The second nucleobase is preferably selected from cytosine and analogs thereof such as 5-azacytosine. It should be noted, however, that also other nucleobases such as 6-methyl purine, 2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-azathymine, 5-fluorouracil, ethyl-4-amino-5-imidazole carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide may be converted to the corresponding deoxyribonucleoside by this nucleobase exchange reaction (Beaussire and Pochet, Nucleosides & Nucleotides 14 (1995), 805-808, Pochet et al. Bioorg. Med. Chem. Lett. 5 (1995), 1679-1684, Pochet and Dugue, Nucleosides & Nucleotides 17 (1998), 2003-2009, Pistotnik et al., Anal. Biochem. 271 (1999), 192-199). This reaction is preferably catalyzed by an enzyme called nucleoside 2-deoxyribosyltransferase (NdT, EC 2.4.2.6) which transfers the glycosyl moiety from a first deoxynucleoside to a second nucleobase, e.g. cytosine. A preferred source of native or recombinant NdT are prokaryotic organisms such as lactobacilli, particularly Lactobacillus leichmannii. Recombinant NdT may be isolated from recombinant E. coli strain pHSP 292 (CNCM I-2191) deposited on Apr. 23, 1999, which is transformed with a plasmid containing the L. leichmannii NdT (nucleoside 2-deoxyribosyltransferase) insert. The nucleotide sequence of the NdT gene and the corresponding amino acid sequence are shown in SEQ ID NO.13 and 14.
[0029]A further aspect of the present invention is a method for the in vitro enzymatic synthesis of deoxyribonucleosides comprising the steps of: (i) condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde to deoxyribose 5-phosphate (dR5P), (ii) isomerizing deoxyribose 5-phosphate to deoxyribose 1-phosphate (dRIP) and (iii) reacting deoxyribose 1-phosphate and a nucleobase, wherein a deoxyribonucleoside and inorganic phosphate are formed. Preferably, the reaction is carried out without isolating intermediate products and, more preferably, as a one-pot reaction. Further, the removal of the inorganic phosphate from the reaction is preferred.
[0030]As outlined above, the glyceraldehyde 3-phosphate may be generated from FDP, DHA and/or GP. Preferably, FDP is used as a starting material.
[0031]In order to avoid the production of undesired by-products and the toxic effects of acetaldehyde, the course of the reaction is preferably controlled by suitable means. Thus, preferably, the reaction is carried out in a manner such that the acetaldehyde concentration in step (ii) is comparatively low, e.g. less than 100 mM, particularly less than 50 mM, e.g. by adding the acetaldehyde in portions or continuously during the course of the reaction and/or by removing excess acetaldehyde. Further, it is preferred that before step (ii) excess starting materials and/or by-products, particularly fructose 1,6-diphosphate and/or deoxyxylulose 1-phosphate (dX1P), are removed. This removal may be effected by chemical and/or enzymatic methods, e.g. precipitating FDP with ferric salts or enzymatically degrading X1P via dihydroxyacetone phosphate. Alternatively or additionally the reaction conditions may be adjusted such that before step (ii) no substantial amounts, preferably less than 10 mM, of starting materials and/or by-products, particularly fructose 1,6-diphosphate and/or deoxyxylulose 1-phosphate, are present in the reaction mixture.
[0032]In still another embodiment, the present invention relates to a method for the in vitro enzymatic synthesis of deoxyribonucleosides comprising the steps of: (i) phosphorylating deoxyribose to deoxyribose 5-phosphate, (ii) isomerizing deoxyribose 5-phosphate to deoxyribose 1-phosphate and (iii) reacting deoxyribose 1-phosphate and nucleobase, wherein a deoxyribonucleoside and inorganic phosphate are formed. Preferably, these reactions are carried out with isolating intermediate products and, more preferably, as a one-pot reaction. To obtain a better yield the removal of inorganic phosphate from step (iii) is preferred.
[0033]By the process as described above naturally occurring deoxyribonucleosides such as dA, dG, dT, dU and dT but also analogs thereof containing non-naturally occurring nucleobases and/or non-naturally occurring deoxyribose sugars such as 2'-deoxy-3'-azido-deoxyribose or 2'-deoxy-4'-thio-deoxy-ribose may be produced.
[0034]The deoxyribonucleosides obtained may be converted to further products according to known methods. These further reaction steps may comprise the synthesis of deoxyribonucleoside mono-, di- or triphosphates, of H-phosphonates or phosphoramidites. Additionally or alternatively, labelling groups such as radioactive or chemical labelling groups may be introduced into the deoxyribonucleosides.
[0035]Still a further aspect of the present invention is the use of an isolated nucleic acid molecule encoding a nucleoside 2-deoxyribosyl transferase (NdT, EC 2.4.2.6) for the preparation of an enzyme in an in vitro enzymatic synthesis process, wherein a deoxyribonucleoside containing a first nucleobase is reacted with a second nucleobase, with a deoxyribonucleoside containing the second nucleobase being formed. The second nucleobase is preferably selected from cytidine and analogs thereof, 2,6-dichloro-purine, 6-chloro-guanine, 6-chloro-purine, 6-aza-thymine and 5-fluoro-uracil. The first nucleobase is preferably selected from thymine, guanine, adenine or uracil. More preferably, the nucleic acid molecule encoding an NdT comprises (a) the nucleotide sequence shown in SEQ ID NO.13 or its complementary sequence, (b) a nucleotide sequence corresponding to the sequence of (a) in the scope of degeneracy of the genetic code or (c) the nucleotide sequence hybridizing under stringent conditions to the sequence (a) and/or (b). Apart from the sequence of SEQ ID NO.13 the present invention also covers nucleotide sequences coding for the same polypeptide, i.e. they correspond to the sequence within the scope of degeneracy of the genetic code, and nucleotide sequence hybridizing with one of the above-mentioned sequences under stringent conditions. These nucleotide sequences are obtainable from SEQ ID NO.13 by recombinant DNA and mutagenesis techniques or from natural sources, e.g. from other Lactobacillus strains.
[0036]Stringent hybridization conditions in the sense of the present invention are defined as those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104. According to this, hybridization under stringent conditions means that a positive hybridization signal is still observed after washing for one hour with 1×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and most preferred at 68° C., in particular, for one hour in 0.2×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and most preferred at 68° C.
[0037]Moreover, the present invention also covers nucleotide sequences which, on nucleotide level, have an identity of at least 70%, particularly preferred at least 80% and most preferred at least 90% to the nucleotide sequence shown in SEQ ID NO.13. Percent identity are determined according to the following equation:
I = n L × 100
wherein I are percent identity, L is the length of the basic sequence and n is the number of nucleotide or amino acid matching between the selected sequence and that of the basic sequence.
[0038]Still another subject matter of the present invention is a recombinant vector comprising at least one copy of the nucleic acid molecule as defined above, operatively linked with an expression control sequence. The vector may be any prokaryotic or eukaryotic vector. Examples of prokaryotic vectors are chromosomal vectors such as bacteriophages (e.g. bacteriophage Lambda) and extrachromosomal vectors such as plasmids (see, for example, Sambrook et al., supra, Chapter 1-4). The vector may also be a eukaryotic vector, e.g. a yeast vector or a vector suitable for higher cells, e.g. a plasmaid vector, viral vector or plant vector. Suitable eukaryotic vectors are described, for example, by Sambrook et al., supra, Chapter 16. The invention moreover relates to a recombinant cell transformed with the nucleic acid or the recombinant vector as described above. The cell may be any cell, e.g. a prokaryotic or eukaryotic cell. Prokaryotic cells, in particular, E. coli cells, are especially preferred.
[0039]The invention refers to an isolated polypeptide having NdT activity encoded by the above-described nucleic acid and its use for the preparation of deoxyribonucleosides. Preferably, the polypeptide has the amino acid sequence shown in SEQ ID NO.14 or an amino acid sequence which is at least 70%, particularly preferred at least 80% and most preferred at least 90% identical thereto, wherein the identity may be determined as described above.
[0040]Finally, the present invention also relates to the use of isolated nucleic acid molecules having thymidine phosphorylase (TP), purine nucleoside phosphorylase (PNP), phosphopentose mutase (PPM), phosphopentose aldolase (PPA), FDP aldolase and deoxyribokinase (dRK) activity for the preparation of an enzyme for a method for the in vitro synthesis of deoxynucleosides. Preferably, these nucleic acids are selected (a) from a nucleotide sequence shown in SEQ ID NO.1, 3, 5, 7, 9 or 11 or their complementary sequences, (b) a nucleotide sequence corresponding to a sequence of (a) within the scope of degeneracy of the genetic code or (c) a nucleotide sequence hybridizing under stringent conditions to a sequence (a) and/or (b).
[0041]Isolated polypeptides having TP, PNP, PPM, PPA, FDP aldolase or dRK activity encoded by the above-described nucleic acids may be used for the preparation of deoxyribonucleosides. Preferably, these polypeptides have the amino acid sequence shown in SEQ ID NO.2, 4, 16, 6, 18, 8, 20, 10 or 12 or an amino acid sequence which is at least 70%, particularly preferred at least 80% and most preferred at least 90% identical thereto, wherein the identity may be determined as described above.
[0042]An isolated nucleic acid molecule encoding a dRK may be used for the preparation of an enzyme for an in vitro method for the enzymatic synthesis of deoxyribonucleosides comprising the step of phosphorylating deoxyribose to deoxyribose 5-phosphate, wherein said nucleic acid molecule comprises (a) the nucleotide sequence shown in SEQ ID NO. 11 or its complementary sequence, (b) a nucleotide sequence corresponding to the sequence of (a) in the scope of the degeneracy of the genetic code or (c) a nucleotide sequence hybridizing under stringent conditions to the sequence of (a) and/or (b). Correspondingly, an isolated polypeptide having dRK activity is suitable for an in vitro method for the enzymatic synthesis of deoxyribonucleosides as outlined above.
[0043]The E. coli strains pHSP 282 (CNCM I-2186), pHSP 283 (CNCM I-2187), pHSP 275 (CNCM I-2188), pHSP 276 (CNCM 2189), pHSP 284 (CNCM I-2190) and pHSP 292 (CNCM I-2191) were deposited according to the regulations of the Budapest Treaty on Apr. 23, 1999 at the Collection Nationale de Culture de Microorganismes, Institut Pasteur, 25, Rue de Docteur Roux, 75724 Paris Cedex 15.
DESCRIPTION OF FIGURES
[0044]FIG. 1 shows the synthesis of dR5P according to Example 12.
[0045]FIG. 2 shows the synthesis of deoxyadenosine according to Example 12.
[0046]FIG. 3 shows the synthesis of deoxyadensine according to Example 13.
[0047]FIG. 4 shows the synthesis of dG-NH2 according to Example 14.
EXAMPLE 1
Sources of Enzymes
[0048]L-glycerol 3-phosphate oxidase (1.1.3.21) from Aerococcus viridans, sucrose phosphorylase (2.4.1.7), fructose 6-phosphate kinase (2.7.1.90) from Propionibacterium freudenreichii, rabbit muscle aldolase (RAMA), formate dehydrogenase, glycerolphosphate dehydrogenase (GDH), triosephosphate isomerase (TIM), catalase, glycerol 3-phosphate oxidase and maltose phosphorylase were obtained from commercial sources (Roche Diagnostics, Sigma) or as described in the literature.
[0049]FDP aldolase II (4.1.2.13), phosphopentose aldolase (PPA, EC 4.1.2.4), phosphopentose mutase (PPM, EC 5.4.2.7), thymidine phosphorylase (TP, EC 2.4.2.4), purine nucleoside phosphorylase (PNP, EC 2.4.2.1), nucleoside 2-deoxyribosyl transferase (NdT, EC 2.4.2.6) were obtained from E. coli strains deposited at CNCM (see above).
EXAMPLE 2
Protocol of the Synthesis of Deoxyadenosine
[0050]Reaction mixture A was prepared by adding acetaldehyde (final concentration 250 mM), FDP aldolase II (0.5 U/ml), PPA (2.5 U/ml) to 20 ml of 100 mM fructose-1,6-diphosphate (FDP), pH 7.6 and incubating overnight at 4° C.
[0051]Mixture B was prepared by adding MnCl2 (final concentration 0.6 mM), glucose 1,6-diphosphate (15 μM), PPM (1.5 U/ml), PNP (0.4 U/ml), SP (1.5 U/ml) pentosephosphate aldolase, PPA (2 U/ml) and FDP aldolase II (0.5 U/ml) to 10 ml 0.9 M sucrose, pH 7.6, at room temperature.
[0052]2 ml of A were added over B at a temperature of 20° C. After 1 hour 2.5 ml A were added. After another hour 3.0 ml A were added. After another 1.5 h 3.5 ml A were added. After another 1.5 h 4 ml A were added and after another 1-1.5 h 5 ml A were added and left to stand overnight.
[0053]At each time of addition of A the amounts of FDP, dR5P, dX1P and dA in the reaction mixture were determined and the yield was calculated. The concentration of acetaldehyde was kept between 20-30 mM. The results are shown in Table 1:
TABLE-US-00001 TABLE 1 Time Volume Concentrations (mM) Yield (mmol) (h) (ml) dR5P dA dX1P dA 0 12 4 0 1.2 0 1 12 3.4 3.2 1 0.04 2 14.5 7.9 8.0 2.6 0.12 3.5 17.5 13 16.2 4.3 0.28 5 21 11.7 21.7 0.46 6 25 23.7 0.59 22 30 11 40.4 13.2 1.21 30 30 50.3 1.51 54 30 8.9 60.6 1.82
[0054]The starting amount of FDP was 1.92 mmol. The amount after completion of reaction was 0.150 mmol. Thus, 1.77 mmol were consumed, theoretically corresponding to 3.54 mmol equivalents dA. The amount of dA formed was 1.82 mmol, leading to a yield of 51.4% based on the amount of FDP.
EXAMPLE 3
Removal of Excess FDP by Means of FeCl3
[0055]1.4 g (2.55 mmol) trisodium-fructose-1,6-disphosphate-octahydrate and 430 μl (335 mg, 7.6 mmol) acetaldehyde were dissolved in 15 ml of water at 4° C. A pH of 7.9 was adjusted by means of sodium hydroxide solution. 150 U pentosephosphate aldolase (PPA) were added, and cold water (4° C.) was added to give 20 ml. After addition of 50 U E. coli aldolase II the mixture was stored at 4° C. After 2 h another 75 U PPA and 50 μl acetaldehyde (390 mg, 8.9 mmol) were added. After 20 h 500 U triosephosphate isomerase (TIM) were added. After 120 h the solution contained about 68 mM FDP, about 12 mM dX1P and about 45 mM dR5P. The reaction was stopped by adding 900 μl of a 2 M solution of iron(III) chloride in 0.01 M hydrochloric acid. The precipitate was centrifuged and washed, the resulting solution contained about 4 mM dX1P, about 9 mM FDP and about 25 mM dR5P.
EXAMPLE 4
Removal of Excess FDP and dX1P by Degradation Via DHAP
[0056]576 mg (1.05 mmol) trisodium-fructose-1,6-disphosphate-octahydrate were dissolved in 8 ml water, and the pH was adjusted at 8.1 by means of sodium hydroxide solution. 75 U PPA and 27 U rabbit muscle aldolase (RAMA) were added, and water was added to give 10 ml. 570 μl (440 mg, 10 mmol) acetaldehyde were added. The reaction was stored at 4° C. After 100 h the solution contained about 110 mM dX1P, about 5 mM FDP and about 85 mM dR5P (about 870 μmol). The reaction was stopped by adding hydrochloric acid until a pH of 2 was reached. After adding sodium hydroxide solution to give a pH of 5.5 the solution was stored.
[0057]For removing dX1P the acetaldehyde was evaporated and the solution was diluted with water to reach 30 ml. It was mixed with 3 ml 2.65 M sodium formate solution (8 mmol), and sodium hydroxide solution was added until a pH of 7.4 was reached. 23 U formate dehydrogenase (FDH), 6 mg NADH, 16 U RAMA and 20 U glycerolphosphate dehydrogenase (GDH) were added.
[0058]After 24 h at room temperature the concentrations of dX1P and FDP are below 3 mM, the loss of dR5P is less than 10%.
EXAMPLE 5
Preparation of dR5P Via G3P
[0059]1.1 g (2.0 mmol) trisodium-fructose-1,6-disphosphate-octahydrate were dissolved in 8 ml water. 1.58 mol of a 2.65 M sodium formate solution (4.2 mmol) and 14.2 mg NADH were added. A pH of 7.0 was adjusted by means of NaOH. After addition of 36 U RAMA, 50 U triosephosphate isomerase (TIM), 34 U GDH and 35 U FDH water was added to give 12 ml.
[0060]After incubation of 40 h at room temperature the FDP content was below 3 mM. The enzymes were denatured by acidification with hydrochloric acid to reach a pH of 2. Subsequently, the pH of the solution was adjusted at 4 and the solids were centrifuged and filtered off, respectively. Through dilution during purification a total volume of 25 ml was reached which contained about 160 mM of glycerol-3-phosphate (G3P).
[0061]4 ml of this solution (about 640 μmol G3P) were adjusted at a pH of 7.8 by means of sodium hydroxide solution. 7.8 kU catalase, 500 U TIM and 13 U glycerol 3-phosphate oxidase are added. The mixture was stirred very slowly in an open flask. After 30 min 18 U PPA were added. Acetaldehyde was added in portions of 30 μl (23.5 mg, 530 μmol) after 30, 60, 120, 180 and 240 min. After 24 h another 15 U PPA, 2.5 kU TIM and 100 μl (78 mg, 1.8 mmol) acetaldehyde were added. After 30 h the batch is sealed after addition of another 100 μl acetaldehyde. After a total of 45 h a concentration of about 60 mM dR5P was achieved and the reaction is completed. For preparing 2'-deoxyadenosine (e.g. Example 7) excess acetaldehyde must be distilled off.
EXAMPLE 6
Preparation of a dR5P Solution Containing Small Amounts of dX1P or FDP
[0062]A solution of 60 mmol/l FDP and 120 mmol/l acetaldehyde having pH 7.4 was kept at a temperature of 15° C. 5 ml thereof were mixed with 4 U aldolase II, 2 U TIM and 40 U PPA and kept at 15° C. After 4, 8.5, 16.5 and 24 h 12 U PPA and 100 μl of a 34 vol.-% solution of acetaldehyde in water (26.4 mg, 600 μmol) were added each. After 40 h the solution was allowed to reach room temperature. After 90 h the reaction solution had reached concentrations of about 3 mM FDP, about 4 mM dX1P and at least 70 mM dR5P. For stopping the reaction and removing acetaldehyde about 20% of the volume were distilled off.
EXAMPLE 7
Preparation of Deoxyadenosine (dA) from dR5P by Means of Barium Acetate
[0063]dR5P was used in the form of a solution prepared according to Examples 3-6. For instance, 10 ml of a solution of Example 6 diluted to have 70 mM dR5P (700 μmol dR5P) were mixed with 40 mg (300 μmol) adenine, 41 μg (50 nmol) tetracyclohexylammonium-glucose-1,6-disphosphate, 396 μg (2 μmol) manganese-II-acetate-tetrahydrate, 10 U pentosephosphate mutase (PPM) and 30 U purine-nucleoside phosphorylase (PNP). After 3 h another 27 mg (200 μmol) adenine and 26 mg (100 μmol) barium acetate were added.
[0064]A further amount of 26 mg barium acetate was added after 4 h, one of 40 mg adenine after 7 h. After 10 h the reaction was completed. The solution had a concentration of 45 mM dA.
EXAMPLE 8
Preparation of Deoxyadenosine (dA) from dR5P by Means of Sucrose Phosphorylase
[0065]10 ml of a solution of Example 6 diluted to 55 mM dR5P (550 μmol dR5P) were mixed with 81 mg (600 μmol) adenine, 41 μq (50 nmol) tetracyclohexylammonium-glucose-1,6-disphosphate, 396 μg (2 μmol) manganese-II-acetate-tetrahydrate, 10 U pentosephosphate mutase (PPM) 15 U purine nucleoside phosphorylase (PNP), 25 U sucrose phosphorylase and 340 mg (1 mmol) cane sugar.
[0066]After 3 h at room temperature the reaction was completed. The solution had a concentration of about 50 mM dA.
EXAMPLE 9
Preparation of Deoxyadenosine (dA) from dR5P by Means of Maltose Phosphorylase
[0067]10 ml of a solution of dR5P diluted to 55 mM were mixed at pH 7.0 with 81 mg (600 μmol) adenine, 41 μg (50 nmoles) glucose 1,6-diphosphate, 396 μg (2 μmoles) manganese II-acetate tetrahydrate, 5 units pentose phosphate mutase (PPM), 10 units purine nucleoside phosphorylase, (PNP), 20 units maltose phosphorylase and 1080 mg (3 mmoles) maltose.
[0068]After 12 h at room temperature the reaction was completed. The solution had a concentration of 49 mM dA.
EXAMPLE 10
Preparation of Deoxycytosine (dC) from dR5P by Means of Sucrose Phosphorylase
[0069]20 ml of a solution of dR5P diluted to 70 mM were mixed at pH 7.0 with 5.4 mg adenine (0.04 mmoles), 155 mg cytosine (1.4 mmoles), 82 μg (100 nmoles) glucose 1,6-diphosphate, 792 μg (4 μmoles) manganese II-acetate-tetrahydrate, 20 units PPM, 30 units PNP, 50 units 2-deoxyribosyl transferase (NdT), 50 units sucrose phosphorylase and 2.05 g (6 mmoles) sucrose.
[0070]After 18 h at 30° C. the solution had a concentration of 62 mM dC.
EXAMPLE 11
Preparation of Deoxyguanosine (dG) from dR5P by Means of Sucrose Phosphorylase
[0071]20 ml of a solution of dR5P diluted to 70 mM were mixed at pH 7.0 with 91 mg guanine (0.6 mmoles), 82 μg (100 nmoles) glucose 1,6-diphosphate, 792 μg (4 μmoles) manganese II-acetate-tetrahydrate, 20 units PPM, 10 units PNP, 20 units sucrose phosphorylase and 2.05 g (6 mmoles) sucrose.
[0072]After 18 h at 37° C. the dG formed corresponds to 0.5 mmoles.
EXAMPLE 12
Two Step Procedure of dA Synthesis
[0073]In the first step dR5P was prepared by adding FDP-Aldolase II (AldII) from E. coli, pentosephosphate aldolase (PPA) from E. coli and triosephosphate isomerase (TIM) from E. coli to fructose-1,6-bisphosphate (FDP) and acetaldehyde (AcAld) essentially according to Ex. 6. FDP trisodium salt was mixed in a final concentration of 75 mM with AcAld (100 mM final concentration). The pH was adjusted to 7.4 by addition of sodium hydroxide. The reaction was started by adding PPA (12 kU/l), Ald II (0.3 kU/l) and TIM (2.5 kU/l). At 4 h 117 mM AcAld, at 7 h 117 mm AcAld, PPA 6 kU/l, TIM 2.5 kU/l and at 12 h 117 mM AcAld were added. The reaction was run at 21° C. Conversion was monitored by enzymatical assay using step by step glycerol-3-phosphate dehydrogenase (GDH), rabbit muscle aldolase (RAMA), trisosephosphate isomerase (TIM), pentosephosphate aldolase (PPA) in the presence of NADH (0.26 mM in 300 mM triethanol amine buffer pH 7.6). Conversion is shown in FIG. 1.
[0074]After yielding approx. 95 mM dR5P the enzymes were deactivated by heating to 65° C. for 10 min. and excess of AcAld was removed by evaporation. In the second step dR5P in a final concentration of 64 mM was converted to deoxyadenosine (dA) by adding adenine (A, final concentration 58 mM) in the presence of 300 μM MnCl2, 5 μM Glucose-1.6-bisphosphate, pentosephosphate mutase from E. coli (PPM, 2 kU/l), purine nucleoside phosphorylase from E. coli (PNP, 1 kU/l). The synthesis was run at 20° C., pH 7.4. In one experiment 200 mM sucrose and 0.6 kU/l sucrose phosphorylase (SP) from Leuconostoc mes. were added at t=2 h (see arrow in FIG. 2, rhombus, solid line), in a second experiment addition of SP was omitted (squares, dotted line). The conversion was monitored by RP-HPLC (column Hypersil ODS 5 μm, 250×4.6 mm; eluent: 30 mM potassium phosphate, 5 mM tetrabutyl ammoniumhydrogensulfate pH 6.0/1% acetonitrile, flow rate: 1 ml/min, column temp.: 35° C., det.: UV at 260 nm) and is shown in FIG. 2.
EXAMPLE 13
[0075]dR5P was prepared by adding FDP-Aldolase II (AldII) from E. coli, pentosephosphate aldolase (PPA) from E. coli and trisosephosphate isomerase (TIM) from E. coli to fructose-1,6-bisphosphate (FDP) and acetaldehyde (AcAld) essentially according to Ex. 6. Excess of AcAld was removed by evaporation. dR5P in a final concentration of 60 mM was converted to deoxyadenosine (dA) by adding adenine (A, final concentration 58 mM) in the presence of 300 μM MnCl2, 5 μM Glucose-1.6-bisphosphate, pentosephosphate mutase from E. coli (PPM, 1.5 kU/l), purine nucleoside phosphorylase from E. coli (PNP, 1 kU/l). The synthesis was run at 20° C., pH 7.4. After 24 h sucrose in a final concentration of 200 mM and sucrose phosphorylase from Leuconsotoc mes. (1 kU/l) were added. Conversion was monitored by RP-HPLC (dA, A, see ex. 12) resp. enzymatical assay (dR5P, using step by step glycerol-3-phosphate dehydrogenase (GDH), rabbit muscle aldolase (RAMA), trisosephosphate isomerase (TIM), pentosephosphate aldolase (PPA) in the presence of NADH (0.26 mM in 300 mM Triethanol amine buffer pH 7.6) and phosphomolybdate complexing of inorg. phosphate (Sigma, Proc. No. 360-UV). This is shown in FIG. 3.
EXAMPLE 14
[0076]dR5P was essentially prepared according to Ex. 6. dR5P in a final concentration of 80 mM was then converted to deoxy-6-aminoguanosine (dG-NH2) by adding 2,6-Diaminopurine (DAP, final concentration 77 mM) in the presence of 200 mM sucrose, 300 μM MnCl2, 5 μM Glucose-1.6-bisphosphate, pentosephosphate mutase from E. coli (PPM, 2.5 kU/l), purine nucleoside phosphorylase from E. coli (PNP, 1 kU/l), sucrose phosphorylase from Leucoonostoc mes. (SP, 1.5 kU/l). The synthesis was run at 20° C. pH 7.4. After 2.5 h, 5 h and 20.5 h additional amounts of enzymes were added: 2.5 h PPM (2.5 kU/l), PNP (1 kU/l, SP (1.5 kU/l), 5 h PPM (2.5 kU/l), SP (1.5 kU/l), 20.5 h: PPM (2.5 kU/l), SP (1.5 kU/l). The conversion was monitored by RP-HPLC (column Hypersil ODS 5 μm, 250×4.6 mm; eluent: 30 mM potassium phosphate, 5 mM tetrabutyl ammoniumhydrogensulfate pH 6.0/1% acetonitrile, flow rate: 1 ml/min, column temp.: 35° C., det.: UV at 216 nm) and is shown in FIG. 4.
Sequence CWU
1
2011323DNAEscherichia coliCDS(1)..(1320) 1ttg ttt ctc gca caa gaa att att
cgt aaa aaa cgt gat ggt cat gcg 48Leu Phe Leu Ala Gln Glu Ile Ile
Arg Lys Lys Arg Asp Gly His Ala 1 5 10
15ctg agc gat gaa gaa att cgt ttc ttt atc aac ggt att cgc
gac aac 96Leu Ser Asp Glu Glu Ile Arg Phe Phe Ile Asn Gly Ile Arg
Asp Asn 20 25 30act atc tcc
gaa ggg cag att gcc gcc ctc gcg atg acc att ttc ttc 144Thr Ile Ser
Glu Gly Gln Ile Ala Ala Leu Ala Met Thr Ile Phe Phe 35
40 45cac gat atg aca atg cct gag cgt gtc tcg ctg
acc atg gcg atg cga 192His Asp Met Thr Met Pro Glu Arg Val Ser Leu
Thr Met Ala Met Arg 50 55 60gat tca
gga acc gtt ctc gac tgg aaa agc ctg cat ctg aat ggc ccg 240Asp Ser
Gly Thr Val Leu Asp Trp Lys Ser Leu His Leu Asn Gly Pro 65
70 75 80att gtt gat aaa cac tcc acc
ggt ggc gtc ggc gat gtg act tcg ctg 288Ile Val Asp Lys His Ser Thr
Gly Gly Val Gly Asp Val Thr Ser Leu 85
90 95atg ttg ggg ccg atg gtc gca gcc tgc ggc ggc tat att
ccg atg atc 336Met Leu Gly Pro Met Val Ala Ala Cys Gly Gly Tyr Ile
Pro Met Ile 100 105 110tct ggt
cgc ggc ctc ggt cat act ggc ggt acg ctc gac aaa ctg gaa 384Ser Gly
Arg Gly Leu Gly His Thr Gly Gly Thr Leu Asp Lys Leu Glu 115
120 125tcc atc cct ggc ttc gac att ttc ccg gat
gac aac cgt ttc cgc gaa 432Ser Ile Pro Gly Phe Asp Ile Phe Pro Asp
Asp Asn Arg Phe Arg Glu 130 135 140att
att aaa gac gtc ggc gtg gcg att atc ggt cag acc agt tca ctg 480Ile
Ile Lys Asp Val Gly Val Ala Ile Ile Gly Gln Thr Ser Ser Leu145
150 155 160gct ccg gct gat aaa cgt
ttc tac gcg acc cgt gat att acc gca acc 528Ala Pro Ala Asp Lys Arg
Phe Tyr Ala Thr Arg Asp Ile Thr Ala Thr 165
170 175gtg gac tcc atc ccg ctg atc acc gcc tct att ctg
gcg aag aaa ctt 576Val Asp Ser Ile Pro Leu Ile Thr Ala Ser Ile Leu
Ala Lys Lys Leu 180 185 190gcg
gaa ggt ctg gac gcg ctg gtg atg gac gtg aaa gtg ggt agc ggc 624Ala
Glu Gly Leu Asp Ala Leu Val Met Asp Val Lys Val Gly Ser Gly 195
200 205gcg ttt atg ccg acc tac gaa ctc tct
gaa gcc ctt gcc gaa gcg att 672Ala Phe Met Pro Thr Tyr Glu Leu Ser
Glu Ala Leu Ala Glu Ala Ile 210 215
220gtt ggc gtg gct aac ggc gct ggc gtg cgc acc acc gcg ctg ctc acc
720Val Gly Val Ala Asn Gly Ala Gly Val Arg Thr Thr Ala Leu Leu Thr225
230 235 240gac atg aat cag
gta ctg gcc tcc agt gca ggt aac gcg gtt gaa gtt 768Asp Met Asn Gln
Val Leu Ala Ser Ser Ala Gly Asn Ala Val Glu Val 245
250 255cgt gaa gcg gtg cag ttc ctg acg ggt gaa
tat cgt aac ccg cgt ctg 816Arg Glu Ala Val Gln Phe Leu Thr Gly Glu
Tyr Arg Asn Pro Arg Leu 260 265
270ttt gat gtc acg atg gcg ctg tgc gtg gag atg ctg atc tcc ggc aaa
864Phe Asp Val Thr Met Ala Leu Cys Val Glu Met Leu Ile Ser Gly Lys
275 280 285ctg gcg aaa gat gac gcc gaa
gcg cgc gcg aaa ttg cag gcg gtg ctg 912Leu Ala Lys Asp Asp Ala Glu
Ala Arg Ala Lys Leu Gln Ala Val Leu 290 295
300gac aac ggt aaa gcg gca gaa gtc ttt ggt cgt atg gta gcg gca caa
960Asp Asn Gly Lys Ala Ala Glu Val Phe Gly Arg Met Val Ala Ala Gln305
310 315 320aaa ggc ccg acc
gac ttc gtt gag aac tac gcg aag tat ctg ccg aca 1008Lys Gly Pro Thr
Asp Phe Val Glu Asn Tyr Ala Lys Tyr Leu Pro Thr 325
330 335gcg atg ctg acg aaa gca gtc tat gct gat
acc gaa ggt ttt gtc agt 1056Ala Met Leu Thr Lys Ala Val Tyr Ala Asp
Thr Glu Gly Phe Val Ser 340 345
350gaa atg gat acc cgc gcg ctg ggg atg gca gtg gtt gca atg ggc ggc
1104Glu Met Asp Thr Arg Ala Leu Gly Met Ala Val Val Ala Met Gly Gly
355 360 365gga cgc cgt cag gca tct gac
acc atc gat tac agc gtc ggc ttt act 1152Gly Arg Arg Gln Ala Ser Asp
Thr Ile Asp Tyr Ser Val Gly Phe Thr 370 375
380gat atg gcg cgt ctg ggc gac cag gta gac ggt cag cgt ccg ctg gcg
1200Asp Met Ala Arg Leu Gly Asp Gln Val Asp Gly Gln Arg Pro Leu Ala385
390 395 400gtt atc cac gcg
aaa gac gaa aac aac tgg cag gaa gcg gcg aaa gcg 1248Val Ile His Ala
Lys Asp Glu Asn Asn Trp Gln Glu Ala Ala Lys Ala 405
410 415gtg aaa gcg gca att aaa ctt gcc gat aaa
gca ccg gaa agc aca cca 1296Val Lys Ala Ala Ile Lys Leu Ala Asp Lys
Ala Pro Glu Ser Thr Pro 420 425
430act gtc tat cgc cgt atc agc gaa taa
1323Thr Val Tyr Arg Arg Ile Ser Glu 435
4402440PRTEscherichia coli 2Leu Phe Leu Ala Gln Glu Ile Ile Arg Lys Lys
Arg Asp Gly His Ala 1 5 10
15Leu Ser Asp Glu Glu Ile Arg Phe Phe Ile Asn Gly Ile Arg Asp Asn
20 25 30Thr Ile Ser Glu Gly Gln
Ile Ala Ala Leu Ala Met Thr Ile Phe Phe 35 40
45His Asp Met Thr Met Pro Glu Arg Val Ser Leu Thr Met Ala
Met Arg 50 55 60Asp Ser Gly Thr Val
Leu Asp Trp Lys Ser Leu His Leu Asn Gly Pro 65 70
75 80Ile Val Asp Lys His Ser Thr Gly Gly Val
Gly Asp Val Thr Ser Leu 85 90
95Met Leu Gly Pro Met Val Ala Ala Cys Gly Gly Tyr Ile Pro Met Ile
100 105 110Ser Gly Arg Gly Leu
Gly His Thr Gly Gly Thr Leu Asp Lys Leu Glu 115
120 125Ser Ile Pro Gly Phe Asp Ile Phe Pro Asp Asp Asn
Arg Phe Arg Glu 130 135 140Ile Ile Lys
Asp Val Gly Val Ala Ile Ile Gly Gln Thr Ser Ser Leu145
150 155 160Ala Pro Ala Asp Lys Arg Phe
Tyr Ala Thr Arg Asp Ile Thr Ala Thr 165
170 175Val Asp Ser Ile Pro Leu Ile Thr Ala Ser Ile Leu
Ala Lys Lys Leu 180 185 190Ala
Glu Gly Leu Asp Ala Leu Val Met Asp Val Lys Val Gly Ser Gly 195
200 205Ala Phe Met Pro Thr Tyr Glu Leu Ser
Glu Ala Leu Ala Glu Ala Ile 210 215
220Val Gly Val Ala Asn Gly Ala Gly Val Arg Thr Thr Ala Leu Leu Thr225
230 235 240Asp Met Asn Gln
Val Leu Ala Ser Ser Ala Gly Asn Ala Val Glu Val 245
250 255Arg Glu Ala Val Gln Phe Leu Thr Gly Glu
Tyr Arg Asn Pro Arg Leu 260 265
270Phe Asp Val Thr Met Ala Leu Cys Val Glu Met Leu Ile Ser Gly Lys
275 280 285Leu Ala Lys Asp Asp Ala Glu
Ala Arg Ala Lys Leu Gln Ala Val Leu 290 295
300Asp Asn Gly Lys Ala Ala Glu Val Phe Gly Arg Met Val Ala Ala
Gln305 310 315 320Lys Gly
Pro Thr Asp Phe Val Glu Asn Tyr Ala Lys Tyr Leu Pro Thr
325 330 335Ala Met Leu Thr Lys Ala Val
Tyr Ala Asp Thr Glu Gly Phe Val Ser 340 345
350Glu Met Asp Thr Arg Ala Leu Gly Met Ala Val Val Ala Met
Gly Gly 355 360 365Gly Arg Arg Gln
Ala Ser Asp Thr Ile Asp Tyr Ser Val Gly Phe Thr 370
375 380Asp Met Ala Arg Leu Gly Asp Gln Val Asp Gly Gln
Arg Pro Leu Ala385 390 395
400Val Ile His Ala Lys Asp Glu Asn Asn Trp Gln Glu Ala Ala Lys Ala
405 410 415Val Lys Ala Ala Ile
Lys Leu Ala Asp Lys Ala Pro Glu Ser Thr Pro 420
425 430Thr Val Tyr Arg Arg Ile Ser Glu 435
4403720DNAEscherichia coliCDS(1)..(717) 3atg gct acc cca cac att
aat gca gaa atg ggc gat ttc gct gac gta 48Met Ala Thr Pro His Ile
Asn Ala Glu Met Gly Asp Phe Ala Asp Val 1 5
10 15gtt ttg atg cca ggc gac ccg ctg cgt gcg aag tat
att gct gaa act 96Val Leu Met Pro Gly Asp Pro Leu Arg Ala Lys Tyr
Ile Ala Glu Thr 20 25 30ttc
ctt gaa gat gcc cgt gaa gtg aac aac gtt cgc ggt atg ctg ggc 144Phe
Leu Glu Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met Leu Gly 35
40 45ttc acc ggt act tac aaa ggc cgc aaa
att tcc gta atg ggt cac ggt 192Phe Thr Gly Thr Tyr Lys Gly Arg Lys
Ile Ser Val Met Gly His Gly 50 55
60atg ggt atc ccg tcc tgc tcc atc tac acc aaa gaa ctg atc acc gat
240Met Gly Ile Pro Ser Cys Ser Ile Tyr Thr Lys Glu Leu Ile Thr Asp 65
70 75 80ttc ggc gtg aag
aaa att atc cgc gtg ggt tcc tgt ggc gca gtt ctg 288Phe Gly Val Lys
Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Leu 85
90 95ccg cac gta aaa ctg cgc gac gtc gtt atc
ggt atg ggt gcc tgc acc 336Pro His Val Lys Leu Arg Asp Val Val Ile
Gly Met Gly Ala Cys Thr 100 105
110gat tcc aaa gtt aac cgc atc cgt ttt aaa gac cat gac ttt gcc gct
384Asp Ser Lys Val Asn Arg Ile Arg Phe Lys Asp His Asp Phe Ala Ala
115 120 125atc gct gac ttc gac atg gtg
cgt aac gca gta gat gca gct aaa gca 432Ile Ala Asp Phe Asp Met Val
Arg Asn Ala Val Asp Ala Ala Lys Ala 130 135
140ctg ggt att gat gct cgc gtg ggt aac ctg ttc tcc gct gac ctg ttc
480Leu Gly Ile Asp Ala Arg Val Gly Asn Leu Phe Ser Ala Asp Leu Phe145
150 155 160tac tct ccg gac
ggc gaa atg ttc gac gtg atg gaa aaa tac ggc att 528Tyr Ser Pro Asp
Gly Glu Met Phe Asp Val Met Glu Lys Tyr Gly Ile 165
170 175ctc ggc gtg gaa atg gaa gcg gct ggt atc
tac ggc gtc gct gca gaa 576Leu Gly Val Glu Met Glu Ala Ala Gly Ile
Tyr Gly Val Ala Ala Glu 180 185
190ttt ggc gcg aaa gcc ctg acc atc tgc acc gta tct gac cac atc cgc
624Phe Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser Asp His Ile Arg
195 200 205act cac gag cag acc act gcc
gct gag cgt cag act acc ttc aac gac 672Thr His Glu Gln Thr Thr Ala
Ala Glu Arg Gln Thr Thr Phe Asn Asp 210 215
220atg atc aaa atc gca ctg gaa tcc gtt ctg ctg ggc gat aaa gag taa
720Met Ile Lys Ile Ala Leu Glu Ser Val Leu Leu Gly Asp Lys Glu225
230 2354239PRTEscherichia coli 4Met Ala Thr
Pro His Ile Asn Ala Glu Met Gly Asp Phe Ala Asp Val 1 5
10 15Val Leu Met Pro Gly Asp Pro Leu Arg
Ala Lys Tyr Ile Ala Glu Thr 20 25
30Phe Leu Glu Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met Leu Gly
35 40 45Phe Thr Gly Thr Tyr Lys
Gly Arg Lys Ile Ser Val Met Gly His Gly 50 55
60Met Gly Ile Pro Ser Cys Ser Ile Tyr Thr Lys Glu Leu Ile Thr
Asp 65 70 75 80Phe Gly
Val Lys Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Leu
85 90 95Pro His Val Lys Leu Arg Asp Val
Val Ile Gly Met Gly Ala Cys Thr 100 105
110Asp Ser Lys Val Asn Arg Ile Arg Phe Lys Asp His Asp Phe Ala
Ala 115 120 125Ile Ala Asp Phe Asp
Met Val Arg Asn Ala Val Asp Ala Ala Lys Ala 130 135
140Leu Gly Ile Asp Ala Arg Val Gly Asn Leu Phe Ser Ala Asp
Leu Phe145 150 155 160Tyr
Ser Pro Asp Gly Glu Met Phe Asp Val Met Glu Lys Tyr Gly Ile
165 170 175Leu Gly Val Glu Met Glu Ala
Ala Gly Ile Tyr Gly Val Ala Ala Glu 180 185
190Phe Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser Asp His
Ile Arg 195 200 205Thr His Glu Gln
Thr Thr Ala Ala Glu Arg Gln Thr Thr Phe Asn Asp 210
215 220Met Ile Lys Ile Ala Leu Glu Ser Val Leu Leu Gly
Asp Lys Glu225 230 23551224DNAEscherichia
coliCDS(1)..(1221) 5atg aaa cgt gca ttt att atg gtg ctg gac tca ttc ggc
atc ggc gct 48Met Lys Arg Ala Phe Ile Met Val Leu Asp Ser Phe Gly
Ile Gly Ala 1 5 10 15aca
gaa gat gca gaa cgc ttt ggt gac gtc ggg gct gac acc ctg ggt 96Thr
Glu Asp Ala Glu Arg Phe Gly Asp Val Gly Ala Asp Thr Leu Gly
20 25 30cat atc gca gaa gct tgt gcc aaa
ggc gaa gct gat aac ggt cgt aaa 144His Ile Ala Glu Ala Cys Ala Lys
Gly Glu Ala Asp Asn Gly Arg Lys 35 40
45ggc ccg ctc aat ctg cca aat ctg acc cgt ctg ggg ctg gcg aaa gca
192Gly Pro Leu Asn Leu Pro Asn Leu Thr Arg Leu Gly Leu Ala Lys Ala
50 55 60cac gaa ggt tct acc ggt ttc att
ccg gcg gga atg gac ggc aac gct 240His Glu Gly Ser Thr Gly Phe Ile
Pro Ala Gly Met Asp Gly Asn Ala 65 70
75 80gaa gtt atc ggc gcg tac gca tgg gcg cac gaa atg tca
tcc ggt aaa 288Glu Val Ile Gly Ala Tyr Ala Trp Ala His Glu Met Ser
Ser Gly Lys 85 90 95gat
acc ccg tct ggt cac tgg gaa att gcc ggt gtc ccg gtt ctg ttt 336Asp
Thr Pro Ser Gly His Trp Glu Ile Ala Gly Val Pro Val Leu Phe
100 105 110gag tgg gga tat ttc tcc gat
cac gaa aac agc ttc ccg caa gag ctg 384Glu Trp Gly Tyr Phe Ser Asp
His Glu Asn Ser Phe Pro Gln Glu Leu 115 120
125ctg gat aaa ctg gtc gaa cgc gct aat ctg ccg ggt tac ctc ggt
aac 432Leu Asp Lys Leu Val Glu Arg Ala Asn Leu Pro Gly Tyr Leu Gly
Asn 130 135 140tgc cac tct tcc ggt acg
gtc att ctg gat caa ctg ggc gaa gag cac 480Cys His Ser Ser Gly Thr
Val Ile Leu Asp Gln Leu Gly Glu Glu His145 150
155 160atg aaa acc ggc aag ccg att ttc tat acc tcc
gct gac tcc gtg ttc 528Met Lys Thr Gly Lys Pro Ile Phe Tyr Thr Ser
Ala Asp Ser Val Phe 165 170
175cag att gcc tgc cat gaa gaa act ttc ggt ctg gat aaa ctc tac gaa
576Gln Ile Ala Cys His Glu Glu Thr Phe Gly Leu Asp Lys Leu Tyr Glu
180 185 190ctg tgc gaa atc gcc cgt
gaa gag ctg acc aac ggc ggc tac aat atc 624Leu Cys Glu Ile Ala Arg
Glu Glu Leu Thr Asn Gly Gly Tyr Asn Ile 195 200
205ggt cgt gtt atc gct cgt ccg ttt atc ggc gac aaa gcc ggt
aac ttc 672Gly Arg Val Ile Ala Arg Pro Phe Ile Gly Asp Lys Ala Gly
Asn Phe 210 215 220cag cgt acc ggt aac
cgt cac gac ctg gct gtt gag ccg cca gca ccg 720Gln Arg Thr Gly Asn
Arg His Asp Leu Ala Val Glu Pro Pro Ala Pro225 230
235 240acc gtg ctg cag aaa ctg gtt gat gaa aaa
cac ggc cag gtg gtt tct 768Thr Val Leu Gln Lys Leu Val Asp Glu Lys
His Gly Gln Val Val Ser 245 250
255gtc ggt aaa att gcg gac atc tac gcc aac tgc ggt atc acc aaa aaa
816Val Gly Lys Ile Ala Asp Ile Tyr Ala Asn Cys Gly Ile Thr Lys Lys
260 265 270gtg aaa gcg act ggc ctg
gac gcg ctg ttt gac gcc acc atc aaa gag 864Val Lys Ala Thr Gly Leu
Asp Ala Leu Phe Asp Ala Thr Ile Lys Glu 275 280
285atg aaa gaa gcg ggt gat aac acc atc gtc ttc acc aac ttc
gtt gac 912Met Lys Glu Ala Gly Asp Asn Thr Ile Val Phe Thr Asn Phe
Val Asp 290 295 300ttc gac tct tcc tgg
ggc cac cgt cgc gac gtc gcc ggt tat gcc gcg 960Phe Asp Ser Ser Trp
Gly His Arg Arg Asp Val Ala Gly Tyr Ala Ala305 310
315 320ggt ctg gaa ctg ttc gac cgc cgt ctg ccg
gag ctg atg tct ctg ctg 1008Gly Leu Glu Leu Phe Asp Arg Arg Leu Pro
Glu Leu Met Ser Leu Leu 325 330
335cgc gat gac gac atc ctg atc ctc acc gct gac cac ggt tgc gat ccg
1056Arg Asp Asp Asp Ile Leu Ile Leu Thr Ala Asp His Gly Cys Asp Pro
340 345 350acc tgg acc ggt act gac
cac acg cgt gaa cac att ccg gta ctg gta 1104Thr Trp Thr Gly Thr Asp
His Thr Arg Glu His Ile Pro Val Leu Val 355 360
365tat ggc ccg aaa gta aaa ccg ggc tca ctg ggt cat cgt gaa
acc ttc 1152Tyr Gly Pro Lys Val Lys Pro Gly Ser Leu Gly His Arg Glu
Thr Phe 370 375 380gcg gat atc ggc cag
act ctg gca aaa tat ttt ggt act tct gat atg 1200Ala Asp Ile Gly Gln
Thr Leu Ala Lys Tyr Phe Gly Thr Ser Asp Met385 390
395 400gaa tat ggc aaa gcc atg ttc tga
1224Glu Tyr Gly Lys Ala Met Phe
4056407PRTEscherichia coli 6Met Lys Arg Ala Phe Ile Met Val Leu Asp Ser
Phe Gly Ile Gly Ala 1 5 10
15Thr Glu Asp Ala Glu Arg Phe Gly Asp Val Gly Ala Asp Thr Leu Gly
20 25 30His Ile Ala Glu Ala Cys
Ala Lys Gly Glu Ala Asp Asn Gly Arg Lys 35 40
45Gly Pro Leu Asn Leu Pro Asn Leu Thr Arg Leu Gly Leu Ala
Lys Ala 50 55 60His Glu Gly Ser Thr
Gly Phe Ile Pro Ala Gly Met Asp Gly Asn Ala 65 70
75 80Glu Val Ile Gly Ala Tyr Ala Trp Ala His
Glu Met Ser Ser Gly Lys 85 90
95Asp Thr Pro Ser Gly His Trp Glu Ile Ala Gly Val Pro Val Leu Phe
100 105 110Glu Trp Gly Tyr Phe
Ser Asp His Glu Asn Ser Phe Pro Gln Glu Leu 115
120 125Leu Asp Lys Leu Val Glu Arg Ala Asn Leu Pro Gly
Tyr Leu Gly Asn 130 135 140Cys His Ser
Ser Gly Thr Val Ile Leu Asp Gln Leu Gly Glu Glu His145
150 155 160Met Lys Thr Gly Lys Pro Ile
Phe Tyr Thr Ser Ala Asp Ser Val Phe 165
170 175Gln Ile Ala Cys His Glu Glu Thr Phe Gly Leu Asp
Lys Leu Tyr Glu 180 185 190Leu
Cys Glu Ile Ala Arg Glu Glu Leu Thr Asn Gly Gly Tyr Asn Ile 195
200 205Gly Arg Val Ile Ala Arg Pro Phe Ile
Gly Asp Lys Ala Gly Asn Phe 210 215
220Gln Arg Thr Gly Asn Arg His Asp Leu Ala Val Glu Pro Pro Ala Pro225
230 235 240Thr Val Leu Gln
Lys Leu Val Asp Glu Lys His Gly Gln Val Val Ser 245
250 255Val Gly Lys Ile Ala Asp Ile Tyr Ala Asn
Cys Gly Ile Thr Lys Lys 260 265
270Val Lys Ala Thr Gly Leu Asp Ala Leu Phe Asp Ala Thr Ile Lys Glu
275 280 285Met Lys Glu Ala Gly Asp Asn
Thr Ile Val Phe Thr Asn Phe Val Asp 290 295
300Phe Asp Ser Ser Trp Gly His Arg Arg Asp Val Ala Gly Tyr Ala
Ala305 310 315 320Gly Leu
Glu Leu Phe Asp Arg Arg Leu Pro Glu Leu Met Ser Leu Leu
325 330 335Arg Asp Asp Asp Ile Leu Ile
Leu Thr Ala Asp His Gly Cys Asp Pro 340 345
350Thr Trp Thr Gly Thr Asp His Thr Arg Glu His Ile Pro Val
Leu Val 355 360 365Tyr Gly Pro Lys
Val Lys Pro Gly Ser Leu Gly His Arg Glu Thr Phe 370
375 380Ala Asp Ile Gly Gln Thr Leu Ala Lys Tyr Phe Gly
Thr Ser Asp Met385 390 395
400Glu Tyr Gly Lys Ala Met Phe 4057780DNAEscherichia
coliCDS(1)..(777) 7atg act gat ctg aaa gca agc agc ctg cgt gca ctg aaa
ttg atg gac 48Met Thr Asp Leu Lys Ala Ser Ser Leu Arg Ala Leu Lys
Leu Met Asp 1 5 10 15ctg
aac acc ctg aat gac gac gac acc gac gag aaa gtg atc gcc ctg 96Leu
Asn Thr Leu Asn Asp Asp Asp Thr Asp Glu Lys Val Ile Ala Leu
20 25 30tgt cat cag gcc aaa act ccg gtc
ggc aat acc gcc gct atc tgt atc 144Cys His Gln Ala Lys Thr Pro Val
Gly Asn Thr Ala Ala Ile Cys Ile 35 40
45tat cct cgc ttt atc ccg att gct cgc aaa act ctg aaa gag cag ggc
192Tyr Pro Arg Phe Ile Pro Ile Ala Arg Lys Thr Leu Lys Glu Gln Gly
50 55 60acc ccg gaa atc cgt atc gct acg
gta acc aac ttc cca cac ggt aac 240Thr Pro Glu Ile Arg Ile Ala Thr
Val Thr Asn Phe Pro His Gly Asn 65 70
75 80gac gac atc gac atc gcg ctg gca gaa acc cgt gcg gca
atc gcc tac 288Asp Asp Ile Asp Ile Ala Leu Ala Glu Thr Arg Ala Ala
Ile Ala Tyr 85 90 95ggt
gct gat gaa gtt gac gtt gtg ttc ccg tac cgc gcg ctg atg gcg 336Gly
Ala Asp Glu Val Asp Val Val Phe Pro Tyr Arg Ala Leu Met Ala
100 105 110ggt aac gag cag gtt ggt ttt
gac ctg gtg aaa gcc tgt aaa gag gct 384Gly Asn Glu Gln Val Gly Phe
Asp Leu Val Lys Ala Cys Lys Glu Ala 115 120
125tgc gcg gca gcg aat gta ctg ctg aaa gtg atc atc gaa acc ggc
gaa 432Cys Ala Ala Ala Asn Val Leu Leu Lys Val Ile Ile Glu Thr Gly
Glu 130 135 140ctg aaa gac gaa gcg ctg
atc cgt aaa gcg tct gaa atc tcc atc aaa 480Leu Lys Asp Glu Ala Leu
Ile Arg Lys Ala Ser Glu Ile Ser Ile Lys145 150
155 160gcg ggt gcg gac ttc atc aaa acc tct acc ggt
aaa gtg gct gtg aac 528Ala Gly Ala Asp Phe Ile Lys Thr Ser Thr Gly
Lys Val Ala Val Asn 165 170
175gcg acg ccg gaa agc gcg cgc atc atg atg gaa gtg atc cgt gat atg
576Ala Thr Pro Glu Ser Ala Arg Ile Met Met Glu Val Ile Arg Asp Met
180 185 190ggc gta gaa aaa acc gtt
ggt ttc aaa ccg gcg ggc ggc gtg cgt act 624Gly Val Glu Lys Thr Val
Gly Phe Lys Pro Ala Gly Gly Val Arg Thr 195 200
205gcg gaa gat gcg cag aaa tat ctc gcc att gca gat gaa ctg
ttc ggt 672Ala Glu Asp Ala Gln Lys Tyr Leu Ala Ile Ala Asp Glu Leu
Phe Gly 210 215 220gct gac tgg gca gat
gcg cgt cac tac cgc ttt ggc gct tcc agc ctg 720Ala Asp Trp Ala Asp
Ala Arg His Tyr Arg Phe Gly Ala Ser Ser Leu225 230
235 240ctg gca agc ctg ctg aaa gcg ctg ggt cac
ggc gac ggt aag agc gcc 768Leu Ala Ser Leu Leu Lys Ala Leu Gly His
Gly Asp Gly Lys Ser Ala 245 250
255agc agc tac taa
780Ser Ser Tyr8259PRTEscherichia coli 8Met Thr Asp Leu Lys Ala Ser Ser
Leu Arg Ala Leu Lys Leu Met Asp 1 5 10
15Leu Asn Thr Leu Asn Asp Asp Asp Thr Asp Glu Lys Val Ile
Ala Leu 20 25 30Cys His Gln
Ala Lys Thr Pro Val Gly Asn Thr Ala Ala Ile Cys Ile 35
40 45Tyr Pro Arg Phe Ile Pro Ile Ala Arg Lys Thr
Leu Lys Glu Gln Gly 50 55 60Thr Pro
Glu Ile Arg Ile Ala Thr Val Thr Asn Phe Pro His Gly Asn 65
70 75 80Asp Asp Ile Asp Ile Ala Leu
Ala Glu Thr Arg Ala Ala Ile Ala Tyr 85
90 95Gly Ala Asp Glu Val Asp Val Val Phe Pro Tyr Arg Ala
Leu Met Ala 100 105 110Gly Asn
Glu Gln Val Gly Phe Asp Leu Val Lys Ala Cys Lys Glu Ala 115
120 125Cys Ala Ala Ala Asn Val Leu Leu Lys Val
Ile Ile Glu Thr Gly Glu 130 135 140Leu
Lys Asp Glu Ala Leu Ile Arg Lys Ala Ser Glu Ile Ser Ile Lys145
150 155 160Ala Gly Ala Asp Phe Ile
Lys Thr Ser Thr Gly Lys Val Ala Val Asn 165
170 175Ala Thr Pro Glu Ser Ala Arg Ile Met Met Glu Val
Ile Arg Asp Met 180 185 190Gly
Val Glu Lys Thr Val Gly Phe Lys Pro Ala Gly Gly Val Arg Thr 195
200 205Ala Glu Asp Ala Gln Lys Tyr Leu Ala
Ile Ala Asp Glu Leu Phe Gly 210 215
220Ala Asp Trp Ala Asp Ala Arg His Tyr Arg Phe Gly Ala Ser Ser Leu225
230 235 240Leu Ala Ser Leu
Leu Lys Ala Leu Gly His Gly Asp Gly Lys Ser Ala 245
250 255Ser Ser Tyr91080DNAEscherichia
coliCDS(1)..(1077) 9atg tct aag att ttt gat ttc gta aaa cct ggc gta atc
act ggt gat 48Met Ser Lys Ile Phe Asp Phe Val Lys Pro Gly Val Ile
Thr Gly Asp 1 5 10 15gac
gta cag aaa gtt ttc cag gta gca aaa gaa aac aac ttc gca ctg 96Asp
Val Gln Lys Val Phe Gln Val Ala Lys Glu Asn Asn Phe Ala Leu
20 25 30cca gca gta aac tgc gtc ggt act
gac tcc atc aac gcc gta ctg gaa 144Pro Ala Val Asn Cys Val Gly Thr
Asp Ser Ile Asn Ala Val Leu Glu 35 40
45acc gct gct aaa gtt aaa gcg ccg gtt atc gtt cag ttc tcc aac ggt
192Thr Ala Ala Lys Val Lys Ala Pro Val Ile Val Gln Phe Ser Asn Gly
50 55 60ggt gct tcc ttt atc gct ggt aaa
ggc gtg aaa tct gac gtt ccg cag 240Gly Ala Ser Phe Ile Ala Gly Lys
Gly Val Lys Ser Asp Val Pro Gln 65 70
75 80ggt gct gct atc ctg ggc gcg atc tct ggt gcg cat cac
gtt cac cag 288Gly Ala Ala Ile Leu Gly Ala Ile Ser Gly Ala His His
Val His Gln 85 90 95atg
gct gaa cat tat ggt gtt ccg gtt atc ctg cac act gac cac tgc 336Met
Ala Glu His Tyr Gly Val Pro Val Ile Leu His Thr Asp His Cys
100 105 110gcg aag aaa ctg ctg ccg tgg
atc gac ggt ctg ttg gac gcg ggt gaa 384Ala Lys Lys Leu Leu Pro Trp
Ile Asp Gly Leu Leu Asp Ala Gly Glu 115 120
125aaa cac ttc gca gct acc ggt aag ccg ctg ttc tct tct cac atg
atc 432Lys His Phe Ala Ala Thr Gly Lys Pro Leu Phe Ser Ser His Met
Ile 130 135 140gac ctg tct gaa gaa tct
ctg caa gag aac atc gaa atc tgc tct aaa 480Asp Leu Ser Glu Glu Ser
Leu Gln Glu Asn Ile Glu Ile Cys Ser Lys145 150
155 160tac ctg gag cgc atg tcc aaa atc ggc atg act
ctg gaa atc gaa ctg 528Tyr Leu Glu Arg Met Ser Lys Ile Gly Met Thr
Leu Glu Ile Glu Leu 165 170
175ggt tgc acc ggt ggt gaa gaa gac ggc gtg gac aac agc cac atg gac
576Gly Cys Thr Gly Gly Glu Glu Asp Gly Val Asp Asn Ser His Met Asp
180 185 190gct tct gca ctg tac acc
cag ccg gaa gac gtt gat tac gca tac acc 624Ala Ser Ala Leu Tyr Thr
Gln Pro Glu Asp Val Asp Tyr Ala Tyr Thr 195 200
205gaa ctg agc aaa atc agc ccg cgt ttc acc atc gca gcg tcc
ttc ggt 672Glu Leu Ser Lys Ile Ser Pro Arg Phe Thr Ile Ala Ala Ser
Phe Gly 210 215 220aac gta cac ggt gtt
tac aag ccg ggt aac gtg gtt ctg act ccg acc 720Asn Val His Gly Val
Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr225 230
235 240atc ctg cgt gat tct cag gaa tat gtt tcc
aag aaa cac aac ctg ccg 768Ile Leu Arg Asp Ser Gln Glu Tyr Val Ser
Lys Lys His Asn Leu Pro 245 250
255cac aac agc ctg aac ttc gta ttc cac ggt ggt tcc ggt tct act gct
816His Asn Ser Leu Asn Phe Val Phe His Gly Gly Ser Gly Ser Thr Ala
260 265 270cag gaa atc aaa gac tcc
gta agc tac ggc gta gta aaa atg aac atc 864Gln Glu Ile Lys Asp Ser
Val Ser Tyr Gly Val Val Lys Met Asn Ile 275 280
285gat acc gat acc caa tgg gca acc tgg gaa ggc gtt ctg aac
tac tac 912Asp Thr Asp Thr Gln Trp Ala Thr Trp Glu Gly Val Leu Asn
Tyr Tyr 290 295 300aaa gcg aac gaa gct
tat ctg cag ggt cag ctg ggt aac ccg aaa ggc 960Lys Ala Asn Glu Ala
Tyr Leu Gln Gly Gln Leu Gly Asn Pro Lys Gly305 310
315 320gaa gat cag ccg aac aag aaa tac tac gat
ccg cgc gta tgg ctg cgt 1008Glu Asp Gln Pro Asn Lys Lys Tyr Tyr Asp
Pro Arg Val Trp Leu Arg 325 330
335gcc ggt cag act tcg atg atc gct cgt ctg gag aaa gca ttc cag gaa
1056Ala Gly Gln Thr Ser Met Ile Ala Arg Leu Glu Lys Ala Phe Gln Glu
340 345 350ctg aac gcg atc gac gtt
ctg taa 1080Leu Asn Ala Ile Asp Val
Leu 35510359PRTEscherichia coli 10Met Ser Lys Ile Phe Asp Phe Val
Lys Pro Gly Val Ile Thr Gly Asp 1 5 10
15Asp Val Gln Lys Val Phe Gln Val Ala Lys Glu Asn Asn Phe
Ala Leu 20 25 30Pro Ala Val
Asn Cys Val Gly Thr Asp Ser Ile Asn Ala Val Leu Glu 35
40 45Thr Ala Ala Lys Val Lys Ala Pro Val Ile Val
Gln Phe Ser Asn Gly 50 55 60Gly Ala
Ser Phe Ile Ala Gly Lys Gly Val Lys Ser Asp Val Pro Gln 65
70 75 80Gly Ala Ala Ile Leu Gly Ala
Ile Ser Gly Ala His His Val His Gln 85
90 95Met Ala Glu His Tyr Gly Val Pro Val Ile Leu His Thr
Asp His Cys 100 105 110Ala Lys
Lys Leu Leu Pro Trp Ile Asp Gly Leu Leu Asp Ala Gly Glu 115
120 125Lys His Phe Ala Ala Thr Gly Lys Pro Leu
Phe Ser Ser His Met Ile 130 135 140Asp
Leu Ser Glu Glu Ser Leu Gln Glu Asn Ile Glu Ile Cys Ser Lys145
150 155 160Tyr Leu Glu Arg Met Ser
Lys Ile Gly Met Thr Leu Glu Ile Glu Leu 165
170 175Gly Cys Thr Gly Gly Glu Glu Asp Gly Val Asp Asn
Ser His Met Asp 180 185 190Ala
Ser Ala Leu Tyr Thr Gln Pro Glu Asp Val Asp Tyr Ala Tyr Thr 195
200 205Glu Leu Ser Lys Ile Ser Pro Arg Phe
Thr Ile Ala Ala Ser Phe Gly 210 215
220Asn Val His Gly Val Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr225
230 235 240Ile Leu Arg Asp
Ser Gln Glu Tyr Val Ser Lys Lys His Asn Leu Pro 245
250 255His Asn Ser Leu Asn Phe Val Phe His Gly
Gly Ser Gly Ser Thr Ala 260 265
270Gln Glu Ile Lys Asp Ser Val Ser Tyr Gly Val Val Lys Met Asn Ile
275 280 285Asp Thr Asp Thr Gln Trp Ala
Thr Trp Glu Gly Val Leu Asn Tyr Tyr 290 295
300Lys Ala Asn Glu Ala Tyr Leu Gln Gly Gln Leu Gly Asn Pro Lys
Gly305 310 315 320Glu Asp
Gln Pro Asn Lys Lys Tyr Tyr Asp Pro Arg Val Trp Leu Arg
325 330 335Ala Gly Gln Thr Ser Met Ile
Ala Arg Leu Glu Lys Ala Phe Gln Glu 340 345
350Leu Asn Ala Ile Asp Val Leu 35511921DNASalmonella
typhiCDS(1)..(918) 11atg gat atc gcg gtt att ggc tct aac atg gtg gac ctt
atc acc tac 48Met Asp Ile Ala Val Ile Gly Ser Asn Met Val Asp Leu
Ile Thr Tyr 1 5 10 15acc
aac cag atg ccc aaa gaa ggg gaa act ctg gaa gcg ccg gcg ttt 96Thr
Asn Gln Met Pro Lys Glu Gly Glu Thr Leu Glu Ala Pro Ala Phe
20 25 30aaa atc ggc tgc ggc gga aaa ggg
gcg aac cag gcc gtg gcg gcc gct 144Lys Ile Gly Cys Gly Gly Lys Gly
Ala Asn Gln Ala Val Ala Ala Ala 35 40
45aag ctc aat tca aaa gta ttg atg ttg acc aaa gtg ggc gac gat att
192Lys Leu Asn Ser Lys Val Leu Met Leu Thr Lys Val Gly Asp Asp Ile
50 55 60ttt gcc gac aac acc att cgt aat
ctc gaa tcc tgg ggg atc aat acg 240Phe Ala Asp Asn Thr Ile Arg Asn
Leu Glu Ser Trp Gly Ile Asn Thr 65 70
75 80acg tat gta gaa aaa gta ccg tgt acc agc agc ggc gta
gcg ccg att 288Thr Tyr Val Glu Lys Val Pro Cys Thr Ser Ser Gly Val
Ala Pro Ile 85 90 95ttc
gtc aac gcc aac tcc agc aac agc att ctg atc atc aaa ggc gct 336Phe
Val Asn Ala Asn Ser Ser Asn Ser Ile Leu Ile Ile Lys Gly Ala
100 105 110aac aag ttt ctc tcg ccg gaa
gat atc gat cgc gcg gcg gaa gat tta 384Asn Lys Phe Leu Ser Pro Glu
Asp Ile Asp Arg Ala Ala Glu Asp Leu 115 120
125aaa aaa tgc cag ctt att gtt ctg caa ctg gaa gtt cag ctt gaa
acg 432Lys Lys Cys Gln Leu Ile Val Leu Gln Leu Glu Val Gln Leu Glu
Thr 130 135 140gtt tat cac gca ata gaa
ttt ggc aag aaa cac ggg att gaa gtg tta 480Val Tyr His Ala Ile Glu
Phe Gly Lys Lys His Gly Ile Glu Val Leu145 150
155 160tta aac cct gcg cca gca tta cgg gaa tta gat
atg tct tat gcc tgt 528Leu Asn Pro Ala Pro Ala Leu Arg Glu Leu Asp
Met Ser Tyr Ala Cys 165 170
175aaa tgc gat ttc ttt gta cct aat gaa acc gag ctg gaa ata tta acc
576Lys Cys Asp Phe Phe Val Pro Asn Glu Thr Glu Leu Glu Ile Leu Thr
180 185 190ggt atg cca gtg gat acc
tat gac cat att cgc gca gcg gca cgt tcg 624Gly Met Pro Val Asp Thr
Tyr Asp His Ile Arg Ala Ala Ala Arg Ser 195 200
205ctg gta gat aaa ggg ctg aac aat att att gtc acc atg ggc
gag aaa 672Leu Val Asp Lys Gly Leu Asn Asn Ile Ile Val Thr Met Gly
Glu Lys 210 215 220ggc gcg ctg tgg atg
acg cgt gac cag gaa gtc cat gtt ccg gcg ttt 720Gly Ala Leu Trp Met
Thr Arg Asp Gln Glu Val His Val Pro Ala Phe225 230
235 240aga gtg aac gct gtt gat acc agc ggc gcg
ggc gat gcc ttt atc ggc 768Arg Val Asn Ala Val Asp Thr Ser Gly Ala
Gly Asp Ala Phe Ile Gly 245 250
255tgt ttc gcg cat tac tac gtc cag agc ggg gat gtg gaa gcc gcc atg
816Cys Phe Ala His Tyr Tyr Val Gln Ser Gly Asp Val Glu Ala Ala Met
260 265 270aaa aaa gcc gtc ctc ttt
gcc gct ttc agc gtc acc ggg aaa ggc acc 864Lys Lys Ala Val Leu Phe
Ala Ala Phe Ser Val Thr Gly Lys Gly Thr 275 280
285caa tcc tct tat cca agc att gag caa ttt aat gag tat ctt
tcg ttg 912Gln Ser Ser Tyr Pro Ser Ile Glu Gln Phe Asn Glu Tyr Leu
Ser Leu 290 295 300aac gaa taa
921Asn
Glu30512306PRTSalmonella typhi 12Met Asp Ile Ala Val Ile Gly Ser Asn Met
Val Asp Leu Ile Thr Tyr 1 5 10
15Thr Asn Gln Met Pro Lys Glu Gly Glu Thr Leu Glu Ala Pro Ala Phe
20 25 30Lys Ile Gly Cys Gly
Gly Lys Gly Ala Asn Gln Ala Val Ala Ala Ala 35
40 45Lys Leu Asn Ser Lys Val Leu Met Leu Thr Lys Val Gly
Asp Asp Ile 50 55 60Phe Ala Asp Asn
Thr Ile Arg Asn Leu Glu Ser Trp Gly Ile Asn Thr 65 70
75 80Thr Tyr Val Glu Lys Val Pro Cys Thr
Ser Ser Gly Val Ala Pro Ile 85 90
95Phe Val Asn Ala Asn Ser Ser Asn Ser Ile Leu Ile Ile Lys Gly
Ala 100 105 110Asn Lys Phe Leu
Ser Pro Glu Asp Ile Asp Arg Ala Ala Glu Asp Leu 115
120 125Lys Lys Cys Gln Leu Ile Val Leu Gln Leu Glu Val
Gln Leu Glu Thr 130 135 140Val Tyr His
Ala Ile Glu Phe Gly Lys Lys His Gly Ile Glu Val Leu145
150 155 160Leu Asn Pro Ala Pro Ala Leu
Arg Glu Leu Asp Met Ser Tyr Ala Cys 165
170 175Lys Cys Asp Phe Phe Val Pro Asn Glu Thr Glu Leu
Glu Ile Leu Thr 180 185 190Gly
Met Pro Val Asp Thr Tyr Asp His Ile Arg Ala Ala Ala Arg Ser 195
200 205Leu Val Asp Lys Gly Leu Asn Asn Ile
Ile Val Thr Met Gly Glu Lys 210 215
220Gly Ala Leu Trp Met Thr Arg Asp Gln Glu Val His Val Pro Ala Phe225
230 235 240Arg Val Asn Ala
Val Asp Thr Ser Gly Ala Gly Asp Ala Phe Ile Gly 245
250 255Cys Phe Ala His Tyr Tyr Val Gln Ser Gly
Asp Val Glu Ala Ala Met 260 265
270Lys Lys Ala Val Leu Phe Ala Ala Phe Ser Val Thr Gly Lys Gly Thr
275 280 285Gln Ser Ser Tyr Pro Ser Ile
Glu Gln Phe Asn Glu Tyr Leu Ser Leu 290 295
300Asn Glu30513483DNALactobacillus leichmanniiCDS(10)..(480)
13gtatactaa atg cca aaa aag acg atc tac ttc ggt gcc ggc tgg ttc act
51 Met Pro Lys Lys Thr Ile Tyr Phe Gly Ala Gly Trp Phe Thr
1 5 10gac cgc caa aac aaa gcc tac aag
gaa gcc atg gaa gcc ctc aag gaa 99Asp Arg Gln Asn Lys Ala Tyr Lys
Glu Ala Met Glu Ala Leu Lys Glu 15 20
25 30aac cca acg att gac ctg gaa aac agc tac gtt ccc ctg
gac aac cag 147Asn Pro Thr Ile Asp Leu Glu Asn Ser Tyr Val Pro Leu
Asp Asn Gln 35 40 45tac
aag ggt atc cgg gtt gat gaa cac ccg gaa tac ctg cat gac aag 195Tyr
Lys Gly Ile Arg Val Asp Glu His Pro Glu Tyr Leu His Asp Lys
50 55 60gtt tgg gct acg gcc acc tac aac
aac gac ttg aac ggg atc aag acc 243Val Trp Ala Thr Ala Thr Tyr Asn
Asn Asp Leu Asn Gly Ile Lys Thr 65 70
75aac gac atc atg ctg ggt gtc tac atc cct gac gaa gaa gac gtc ggc
291Asn Asp Ile Met Leu Gly Val Tyr Ile Pro Asp Glu Glu Asp Val Gly
80 85 90ctg ggc atg gaa ctg ggt tac gcc
ttg agc caa ggc aag tac gtc ctt 339Leu Gly Met Glu Leu Gly Tyr Ala
Leu Ser Gln Gly Lys Tyr Val Leu 95 100
105 110ttg gtc atc ccg gac gaa gac tac ggc aag ccg atc
aac ctc atg agc 387Leu Val Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile
Asn Leu Met Ser 115 120
125tgg ggc gtc agc gac aac gtg atc aag atg agc cag ctg aag gac ttc
435Trp Gly Val Ser Asp Asn Val Ile Lys Met Ser Gln Leu Lys Asp Phe
130 135 140aac ttc aac aag ccg cgc
ttc gac ttc tac gaa ggt gcc gta tac taa 483Asn Phe Asn Lys Pro Arg
Phe Asp Phe Tyr Glu Gly Ala Val Tyr 145 150
15514157PRTLactobacillus leichmannii 14Met Pro Lys Lys Thr Ile
Tyr Phe Gly Ala Gly Trp Phe Thr Asp Arg 1 5
10 15Gln Asn Lys Ala Tyr Lys Glu Ala Met Glu Ala Leu
Lys Glu Asn Pro 20 25 30Thr
Ile Asp Leu Glu Asn Ser Tyr Val Pro Leu Asp Asn Gln Tyr Lys 35
40 45Gly Ile Arg Val Asp Glu His Pro Glu
Tyr Leu His Asp Lys Val Trp 50 55
60Ala Thr Ala Thr Tyr Asn Asn Asp Leu Asn Gly Ile Lys Thr Asn Asp 65
70 75 80Ile Met Leu Gly Val
Tyr Ile Pro Asp Glu Glu Asp Val Gly Leu Gly 85
90 95Met Glu Leu Gly Tyr Ala Leu Ser Gln Gly Lys
Tyr Val Leu Leu Val 100 105
110Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile Asn Leu Met Ser Trp Gly
115 120 125Val Ser Asp Asn Val Ile Lys
Met Ser Gln Leu Lys Asp Phe Asn Phe 130 135
140Asn Lys Pro Arg Phe Asp Phe Tyr Glu Gly Ala Val Tyr145
150 15515720DNAEscherichia coliCDS(1)..(717) 15atg
gct acc cca cac att aat gca gaa atg ggc gat ttc gct gac gta 48Met
Ala Thr Pro His Ile Asn Ala Glu Met Gly Asp Phe Ala Asp Val 1
5 10 15gtt ttg atg cca ggc gac ccg
ctg cgt gcg aag tat att gct gaa act 96Val Leu Met Pro Gly Asp Pro
Leu Arg Ala Lys Tyr Ile Ala Glu Thr 20 25
30ttc ctt gaa gat gcc cgt gaa gtg aac aac gtt cgc ggt atg
ctg ggc 144Phe Leu Glu Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met
Leu Gly 35 40 45ttc acc ggt act
tac aaa ggc cgc aaa att tcc gta atg ggt cac ggt 192Phe Thr Gly Thr
Tyr Lys Gly Arg Lys Ile Ser Val Met Gly His Gly 50
55 60atg ggt atc ccg tcc tgc tcc atc tac acc aaa gaa ctg
atc acc gat 240Met Gly Ile Pro Ser Cys Ser Ile Tyr Thr Lys Glu Leu
Ile Thr Asp 65 70 75
80ttc ggc gtg aag aaa att atc cgc gtg ggt tcc tgt ggc gca gtt ctg
288Phe Gly Val Lys Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Leu
85 90 95ccg cac gta aaa ctg
cgc gac gtc gtt atc ggt atg ggt acc tgc acc 336Pro His Val Lys Leu
Arg Asp Val Val Ile Gly Met Gly Thr Cys Thr 100
105 110gat tcc aaa gtt aac cgc atc cgt ttt aaa gac cat
gac ttt gcc gct 384Asp Ser Lys Val Asn Arg Ile Arg Phe Lys Asp His
Asp Phe Ala Ala 115 120 125atc gct
gac ttc gac atg gtg cgt aac gca gta gat gca gct aaa gca 432Ile Ala
Asp Phe Asp Met Val Arg Asn Ala Val Asp Ala Ala Lys Ala 130
135 140ctg ggt att gat gct cgc gtg ggt aac ctg ttc
tcc gct gac ctg ttc 480Leu Gly Ile Asp Ala Arg Val Gly Asn Leu Phe
Ser Ala Asp Leu Phe145 150 155
160tac tct ccg gac ggc gaa atg ttc gac gtg atg gaa aaa tac ggc att
528Tyr Ser Pro Asp Gly Glu Met Phe Asp Val Met Glu Lys Tyr Gly Ile
165 170 175ctc ggc gtg gaa atg
gaa gcg gct ggt atc tac ggc gtc gct gca gaa 576Leu Gly Val Glu Met
Glu Ala Ala Gly Ile Tyr Gly Val Ala Ala Glu 180
185 190ttt ggc gcg aaa gcc ctg acc atc tgc acc gta tct
gac cac atc cgc 624Phe Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser
Asp His Ile Arg 195 200 205act cac
gag cag acc act gcc gct gag cgt cag act acc ttc aac aac 672Thr His
Glu Gln Thr Thr Ala Ala Glu Arg Gln Thr Thr Phe Asn Asn 210
215 220atg atc aaa atc gca ctg gaa tcc gtt ctg ctg
ggc gat aaa gag taa 720Met Ile Lys Ile Ala Leu Glu Ser Val Leu Leu
Gly Asp Lys Glu225 230
23516239PRTEscherichia coli 16Met Ala Thr Pro His Ile Asn Ala Glu Met Gly
Asp Phe Ala Asp Val 1 5 10
15Val Leu Met Pro Gly Asp Pro Leu Arg Ala Lys Tyr Ile Ala Glu Thr
20 25 30Phe Leu Glu Asp Ala Arg
Glu Val Asn Asn Val Arg Gly Met Leu Gly 35 40
45Phe Thr Gly Thr Tyr Lys Gly Arg Lys Ile Ser Val Met Gly
His Gly 50 55 60Met Gly Ile Pro Ser
Cys Ser Ile Tyr Thr Lys Glu Leu Ile Thr Asp 65 70
75 80Phe Gly Val Lys Lys Ile Ile Arg Val Gly
Ser Cys Gly Ala Val Leu 85 90
95Pro His Val Lys Leu Arg Asp Val Val Ile Gly Met Gly Thr Cys Thr
100 105 110Asp Ser Lys Val Asn
Arg Ile Arg Phe Lys Asp His Asp Phe Ala Ala 115
120 125Ile Ala Asp Phe Asp Met Val Arg Asn Ala Val Asp
Ala Ala Lys Ala 130 135 140Leu Gly Ile
Asp Ala Arg Val Gly Asn Leu Phe Ser Ala Asp Leu Phe145
150 155 160Tyr Ser Pro Asp Gly Glu Met
Phe Asp Val Met Glu Lys Tyr Gly Ile 165
170 175Leu Gly Val Glu Met Glu Ala Ala Gly Ile Tyr Gly
Val Ala Ala Glu 180 185 190Phe
Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser Asp His Ile Arg 195
200 205Thr His Glu Gln Thr Thr Ala Ala Glu
Arg Gln Thr Thr Phe Asn Asn 210 215
220Met Ile Lys Ile Ala Leu Glu Ser Val Leu Leu Gly Asp Lys Glu225
230 235171224DNAEscherichia coliCDS(1)..(1221)
17atg aaa cgt gca ttt att atg gtg ctg gac tca ttc ggc atc ggc gct
48Met Lys Arg Ala Phe Ile Met Val Leu Asp Ser Phe Gly Ile Gly Ala 1
5 10 15aca gaa gat gca gaa cgc
ttt ggt gac gtc ggg gct gac acc ctg ggt 96Thr Glu Asp Ala Glu Arg
Phe Gly Asp Val Gly Ala Asp Thr Leu Gly 20
25 30cat atc gca gaa gct tgt gcc aaa ggc gaa gct gat aac
ggt cgt aaa 144His Ile Ala Glu Ala Cys Ala Lys Gly Glu Ala Asp Asn
Gly Arg Lys 35 40 45ggc ccg ctc
aat ctg cca aat ctg acc cgt ctg ggg ctg gcg aaa gca 192Gly Pro Leu
Asn Leu Pro Asn Leu Thr Arg Leu Gly Leu Ala Lys Ala 50
55 60cac gaa ggt tct acc ggt ttc att ccg gcg gga atg
gac ggc aac gct 240His Glu Gly Ser Thr Gly Phe Ile Pro Ala Gly Met
Asp Gly Asn Ala 65 70 75
80gaa gtt atc ggc gcg tac gca tgg gcg cac gaa atg tca tcc ggt aaa
288Glu Val Ile Gly Ala Tyr Ala Trp Ala His Glu Met Ser Ser Gly Lys
85 90 95gat acc ccg tct ggt
cac tgg gaa att gcc ggc gtc ccg gtt ctg ttt 336Asp Thr Pro Ser Gly
His Trp Glu Ile Ala Gly Val Pro Val Leu Phe 100
105 110gag tgg gga tat ttc tcc gat cac gaa aac agc ttc
ccg caa gag ctg 384Glu Trp Gly Tyr Phe Ser Asp His Glu Asn Ser Phe
Pro Gln Glu Leu 115 120 125ctg gat
aaa ctg gtc gaa cgc gct aat ctg ccg ggt tac ctc ggt aac 432Leu Asp
Lys Leu Val Glu Arg Ala Asn Leu Pro Gly Tyr Leu Gly Asn 130
135 140tgc cac tct tcc ggt acg gtc att ctg gat caa
ctg ggc gaa gag cac 480Cys His Ser Ser Gly Thr Val Ile Leu Asp Gln
Leu Gly Glu Glu His145 150 155
160atg aaa acc ggc aag ccg att ttc tat acc tcc gct gac tcc gtg ttc
528Met Lys Thr Gly Lys Pro Ile Phe Tyr Thr Ser Ala Asp Ser Val Phe
165 170 175cag att gcc tgc cat
gaa gaa act ttc ggt ctg gat aaa ctc tac gaa 576Gln Ile Ala Cys His
Glu Glu Thr Phe Gly Leu Asp Lys Leu Tyr Glu 180
185 190ctg tgc gaa atc gcc cgt gaa gag ctg acc aac ggc
ggc tac aat atc 624Leu Cys Glu Ile Ala Arg Glu Glu Leu Thr Asn Gly
Gly Tyr Asn Ile 195 200 205ggt cgt
gtt atc gct cgt ccg ttt atc ggc gac aaa gcc ggt aac ttc 672Gly Arg
Val Ile Ala Arg Pro Phe Ile Gly Asp Lys Ala Gly Asn Phe 210
215 220caa cgt acc ggt aac cgt cac gac ctg gct gtt
gag ccg cca gca ccg 720Gln Arg Thr Gly Asn Arg His Asp Leu Ala Val
Glu Pro Pro Ala Pro225 230 235
240acc gtg ctg cag aaa ctg gtt gat gaa aaa cac ggc cag gtg gtt tct
768Thr Val Leu Gln Lys Leu Val Asp Glu Lys His Gly Gln Val Val Ser
245 250 255gtc ggt aaa att gcg
gac atc tac gcc aac tgc ggt atc acc aaa aaa 816Val Gly Lys Ile Ala
Asp Ile Tyr Ala Asn Cys Gly Ile Thr Lys Lys 260
265 270gtg aaa gcg act ggc ctg gac gcg ctg ttt gac acc
acc atc aaa gag 864Val Lys Ala Thr Gly Leu Asp Ala Leu Phe Asp Thr
Thr Ile Lys Glu 275 280 285atg aaa
gaa gcg ggt gat aac acc atc gtc ttc acc aac ttc gtt gac 912Met Lys
Glu Ala Gly Asp Asn Thr Ile Val Phe Thr Asn Phe Val Asp 290
295 300ttc gac tct tcc tgg ggc cac cgt cgc gac gtc
gcc ggt tat gcc gcg 960Phe Asp Ser Ser Trp Gly His Arg Arg Asp Val
Ala Gly Tyr Ala Ala305 310 315
320ggt ctg gaa ctg ttc gac cgc cgt ctg ccg gag ctg atg tct ctg ctg
1008Gly Leu Glu Leu Phe Asp Arg Arg Leu Pro Glu Leu Met Ser Leu Leu
325 330 335cgc gat gac gac atc
ctg atc ctc acc gct gac cac ggt tgc gat ccg 1056Arg Asp Asp Asp Ile
Leu Ile Leu Thr Ala Asp His Gly Cys Asp Pro 340
345 350acc tgg acc ggt act gac cac acg cgt gaa cac att
ccg gta ctg gta 1104Thr Trp Thr Gly Thr Asp His Thr Arg Glu His Ile
Pro Val Leu Val 355 360 365tat ggc
ccg aaa gta aaa ccg ggc tca ctg ggt cat cgt gaa acc ttc 1152Tyr Gly
Pro Lys Val Lys Pro Gly Ser Leu Gly His Arg Glu Thr Phe 370
375 380gcg gat atc ggc cag act ctg gca aaa tat ttt
ggt act tct gat atg 1200Ala Asp Ile Gly Gln Thr Leu Ala Lys Tyr Phe
Gly Thr Ser Asp Met385 390 395
400gaa tat ggc aaa gcc atg ttc tga
1224Glu Tyr Gly Lys Ala Met Phe 40518407PRTEscherichia
coli 18Met Lys Arg Ala Phe Ile Met Val Leu Asp Ser Phe Gly Ile Gly Ala 1
5 10 15Thr Glu Asp Ala
Glu Arg Phe Gly Asp Val Gly Ala Asp Thr Leu Gly 20
25 30His Ile Ala Glu Ala Cys Ala Lys Gly Glu Ala
Asp Asn Gly Arg Lys 35 40 45Gly
Pro Leu Asn Leu Pro Asn Leu Thr Arg Leu Gly Leu Ala Lys Ala 50
55 60His Glu Gly Ser Thr Gly Phe Ile Pro Ala
Gly Met Asp Gly Asn Ala 65 70 75
80Glu Val Ile Gly Ala Tyr Ala Trp Ala His Glu Met Ser Ser Gly
Lys 85 90 95Asp Thr Pro
Ser Gly His Trp Glu Ile Ala Gly Val Pro Val Leu Phe 100
105 110Glu Trp Gly Tyr Phe Ser Asp His Glu Asn
Ser Phe Pro Gln Glu Leu 115 120
125Leu Asp Lys Leu Val Glu Arg Ala Asn Leu Pro Gly Tyr Leu Gly Asn 130
135 140Cys His Ser Ser Gly Thr Val Ile
Leu Asp Gln Leu Gly Glu Glu His145 150
155 160Met Lys Thr Gly Lys Pro Ile Phe Tyr Thr Ser Ala
Asp Ser Val Phe 165 170
175Gln Ile Ala Cys His Glu Glu Thr Phe Gly Leu Asp Lys Leu Tyr Glu
180 185 190Leu Cys Glu Ile Ala Arg
Glu Glu Leu Thr Asn Gly Gly Tyr Asn Ile 195 200
205Gly Arg Val Ile Ala Arg Pro Phe Ile Gly Asp Lys Ala Gly
Asn Phe 210 215 220Gln Arg Thr Gly Asn
Arg His Asp Leu Ala Val Glu Pro Pro Ala Pro225 230
235 240Thr Val Leu Gln Lys Leu Val Asp Glu Lys
His Gly Gln Val Val Ser 245 250
255Val Gly Lys Ile Ala Asp Ile Tyr Ala Asn Cys Gly Ile Thr Lys Lys
260 265 270Val Lys Ala Thr Gly
Leu Asp Ala Leu Phe Asp Thr Thr Ile Lys Glu 275
280 285Met Lys Glu Ala Gly Asp Asn Thr Ile Val Phe Thr
Asn Phe Val Asp 290 295 300Phe Asp Ser
Ser Trp Gly His Arg Arg Asp Val Ala Gly Tyr Ala Ala305
310 315 320Gly Leu Glu Leu Phe Asp Arg
Arg Leu Pro Glu Leu Met Ser Leu Leu 325
330 335Arg Asp Asp Asp Ile Leu Ile Leu Thr Ala Asp His
Gly Cys Asp Pro 340 345 350Thr
Trp Thr Gly Thr Asp His Thr Arg Glu His Ile Pro Val Leu Val 355
360 365Tyr Gly Pro Lys Val Lys Pro Gly Ser
Leu Gly His Arg Glu Thr Phe 370 375
380Ala Asp Ile Gly Gln Thr Leu Ala Lys Tyr Phe Gly Thr Ser Asp Met385
390 395 400Glu Tyr Gly Lys
Ala Met Phe 40519780DNAEscherichia coliCDS(1)..(777) 19atg
act gat ctg aaa gca agc agc ctg cgt gca ctg aaa ttg atg gac 48Met
Thr Asp Leu Lys Ala Ser Ser Leu Arg Ala Leu Lys Leu Met Asp 1
5 10 15ctg aac acc ctg aat gac gac
gac acc gac gag aaa gtg atc gcc ctg 96Leu Asn Thr Leu Asn Asp Asp
Asp Thr Asp Glu Lys Val Ile Ala Leu 20 25
30tgt cat cag gcc aaa act ccg gtc ggc aat acc gcc gct atc
tgt atc 144Cys His Gln Ala Lys Thr Pro Val Gly Asn Thr Ala Ala Ile
Cys Ile 35 40 45tat cct cgc ttt
atc ccg att gct cgc aaa act ctg aaa gag cag ggc 192Tyr Pro Arg Phe
Ile Pro Ile Ala Arg Lys Thr Leu Lys Glu Gln Gly 50
55 60acc ccg gaa atc cgt atc gct acg gta acc aac ttc cca
cac ggt aac 240Thr Pro Glu Ile Arg Ile Ala Thr Val Thr Asn Phe Pro
His Gly Asn 65 70 75
80gac gac atc gac atc gcg ctg gca gaa acc cgt gcg gca atc gcc tac
288Asp Asp Ile Asp Ile Ala Leu Ala Glu Thr Arg Ala Ala Ile Ala Tyr
85 90 95ggt gct gat gaa gtt
gac gtt gtg ttc ccg tac cgc gcg ctg atg gcg 336Gly Ala Asp Glu Val
Asp Val Val Phe Pro Tyr Arg Ala Leu Met Ala 100
105 110ggt aac gag cag gtt ggt ttt gac ctg gtg aaa gcc
tgt aaa gag gct 384Gly Asn Glu Gln Val Gly Phe Asp Leu Val Lys Ala
Cys Lys Glu Ala 115 120 125tgc gcg
gca gcg aat gta ctg ctg aaa gtg atc atc gaa acc ggc gaa 432Cys Ala
Ala Ala Asn Val Leu Leu Lys Val Ile Ile Glu Thr Gly Glu 130
135 140ctg aaa gac gaa gcg ctg atc cgt aaa gcg tct
gaa atc tcc atc aaa 480Leu Lys Asp Glu Ala Leu Ile Arg Lys Ala Ser
Glu Ile Ser Ile Lys145 150 155
160gcg ggt gtg gac ttc atc aaa acc tct acc ggt aaa gtg gct gtg aac
528Ala Gly Val Asp Phe Ile Lys Thr Ser Thr Gly Lys Val Ala Val Asn
165 170 175gcg acg ccg gaa agc
gcg cgc atc atg atg gaa gtg atc cgt gat atg 576Ala Thr Pro Glu Ser
Ala Arg Ile Met Met Glu Val Ile Arg Asp Met 180
185 190ggc gta gaa aaa acc gtt ggt ttc aaa ccg gcg ggc
ggc gtg cgt act 624Gly Val Glu Lys Thr Val Gly Phe Lys Pro Ala Gly
Gly Val Arg Thr 195 200 205gcg gaa
gat gcg cag aaa tat ctc gcc att gca gat gaa ctg ttc ggt 672Ala Glu
Asp Ala Gln Lys Tyr Leu Ala Ile Ala Asp Glu Leu Phe Gly 210
215 220gct gac tgg gca gat gcg cgt cac tac cgc ttt
ggc gct tcc agc ctg 720Ala Asp Trp Ala Asp Ala Arg His Tyr Arg Phe
Gly Ala Ser Ser Leu225 230 235
240ctg gca agc ctg ctg aaa gcg ctg ggt cac ggc gac ggt aag agc gcc
768Leu Ala Ser Leu Leu Lys Ala Leu Gly His Gly Asp Gly Lys Ser Ala
245 250 255agc agc tac taa
780Ser Ser
Tyr20259PRTEscherichia coli 20Met Thr Asp Leu Lys Ala Ser Ser Leu Arg Ala
Leu Lys Leu Met Asp 1 5 10
15Leu Asn Thr Leu Asn Asp Asp Asp Thr Asp Glu Lys Val Ile Ala Leu
20 25 30Cys His Gln Ala Lys Thr
Pro Val Gly Asn Thr Ala Ala Ile Cys Ile 35 40
45Tyr Pro Arg Phe Ile Pro Ile Ala Arg Lys Thr Leu Lys Glu
Gln Gly 50 55 60Thr Pro Glu Ile Arg
Ile Ala Thr Val Thr Asn Phe Pro His Gly Asn 65 70
75 80Asp Asp Ile Asp Ile Ala Leu Ala Glu Thr
Arg Ala Ala Ile Ala Tyr 85 90
95Gly Ala Asp Glu Val Asp Val Val Phe Pro Tyr Arg Ala Leu Met Ala
100 105 110Gly Asn Glu Gln Val
Gly Phe Asp Leu Val Lys Ala Cys Lys Glu Ala 115
120 125Cys Ala Ala Ala Asn Val Leu Leu Lys Val Ile Ile
Glu Thr Gly Glu 130 135 140Leu Lys Asp
Glu Ala Leu Ile Arg Lys Ala Ser Glu Ile Ser Ile Lys145
150 155 160Ala Gly Val Asp Phe Ile Lys
Thr Ser Thr Gly Lys Val Ala Val Asn 165
170 175Ala Thr Pro Glu Ser Ala Arg Ile Met Met Glu Val
Ile Arg Asp Met 180 185 190Gly
Val Glu Lys Thr Val Gly Phe Lys Pro Ala Gly Gly Val Arg Thr 195
200 205Ala Glu Asp Ala Gln Lys Tyr Leu Ala
Ile Ala Asp Glu Leu Phe Gly 210 215
220Ala Asp Trp Ala Asp Ala Arg His Tyr Arg Phe Gly Ala Ser Ser Leu225
230 235 240Leu Ala Ser Leu
Leu Lys Ala Leu Gly His Gly Asp Gly Lys Ser Ala 245
250 255Ser Ser Tyr
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