Purification and Characterization of CobT, the Nicotinate-mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase Enzyme from Salmonella typhimurium LT2

We report the purification and biochemical characterization of the cobalamin biosynthetic enzyme nicotinate-mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium. cobT was overexpressed and the protein purified to approximately 97% homogeneity. NH2-terminal sequence analysis confirmed that the protein encoded by cobT was purified. Homogeneous CobT catalyzed the synthesis ofN 1-(5-phospho-α-D-ribosyl)-5,6-dimethylbenzimidazole. The identity of high performance liquid chromatography-purified product was confirmed by fast atom bombardment mass spectrometry. CobT activity was optimal at 45 °C and pH 10.0. The apparentK m for nicotinate mononucleotide was 680 μm; the apparent K m for 5,6-dimethylbenzimidazole was less than 10 μm. CobT used nicotinamide mononucleotide as a ribose phosphate donor. CobT phosphoribosylated alternative base substrates including benzimidazole, 4,5-dimethyl-1,2-phenylenediamine, imidazole, histidine, adenine, and guanine in vitro. The resulting ribotides were incorporated into cobamides that were differentially utilized by methionine synthase (EC 2.1.1.13), ethanolamine ammonia-lyase (EC 4.3.1.7), and 1,2-propanediol dehydratase (EC 4.2.1.28) in vivo. The lack of base substrate specificity by CobT may explain the inability to isolate mutants blocked in the synthesis of 5,6-dimethylbenzimidazole in this bacterium. We report the purification and biochemical characterization of the cobalamin biosynthetic enzyme nicotinate-mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium. cobT was overexpressed and the protein purified to approximately 97% homogeneity. NH2-terminal sequence analysis confirmed that the protein encoded by cobT was purified. Homogeneous CobT catalyzed the synthesis ofN 1-(5-phospho-α-D-ribosyl)-5,6-dimethylbenzimidazole. The identity of high performance liquid chromatography-purified product was confirmed by fast atom bombardment mass spectrometry. CobT activity was optimal at 45 °C and pH 10.0. The apparentK m for nicotinate mononucleotide was 680 μm; the apparent K m for 5,6-dimethylbenzimidazole was less than 10 μm. CobT used nicotinamide mononucleotide as a ribose phosphate donor. CobT phosphoribosylated alternative base substrates including benzimidazole, 4,5-dimethyl-1,2-phenylenediamine, imidazole, histidine, adenine, and guanine in vitro. The resulting ribotides were incorporated into cobamides that were differentially utilized by methionine synthase (EC 2.1.1.13), ethanolamine ammonia-lyase (EC 4.3.1.7), and 1,2-propanediol dehydratase (EC 4.2.1.28) in vivo. The lack of base substrate specificity by CobT may explain the inability to isolate mutants blocked in the synthesis of 5,6-dimethylbenzimidazole in this bacterium. The primary cobamide synthesized by Salmonella typhimurium LT2 isCoα-5,6-dimethylbenzimidazolyl-Coβ-adenosylcobamide (Ado-Cbl) 1The abbreviations used are: Ado-Cbl, adenosyl-cobalamin; Me2Bza, 5,6-dimethylbenzimidazole; Bza, benzimidazole; NaMN, nicotinate-mononucleotide; Me2Pda, 4,5-dimethyl-1,2-phenylenediamine; α-ribazole 5′-phosphate,N 1-(5-phospho-α-D-ribosyl)-5,6-dimethylbenzimidazole; NMN, nicotinamide mononucleotide; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: Ado-Cbl, adenosyl-cobalamin; Me2Bza, 5,6-dimethylbenzimidazole; Bza, benzimidazole; NaMN, nicotinate-mononucleotide; Me2Pda, 4,5-dimethyl-1,2-phenylenediamine; α-ribazole 5′-phosphate,N 1-(5-phospho-α-D-ribosyl)-5,6-dimethylbenzimidazole; NMN, nicotinamide mononucleotide; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis. (Fig. 1). Cobamides containing compounds other than 5,6-dimethylbenzimidazole (Me2Bza) as lower (Coα) ligands have been isolated from other prokaryotes. The lower ligands fall within two classes: (i) derivatives or analogs of benzimidazole (Bza) (e.g. 5-hydroxybenzimidazole, 5-methylbenzimidazole, 5-methoxy-6-methylbenzimidazole, adenine) (3Stupperich E. Kräutler B. Arch. Microbiol. 1988; 149: 213-217Crossref Scopus (53) Google Scholar, 4Kräutler B. Kohler H.P.E. Stupperich E. Eur. J. Biochem. 1988; 176: 461-469Crossref PubMed Scopus (33) Google Scholar, 5Stupperich E. Eisinger H.J. Kräutler B. Eur. J. Biochem. 1988; 172: 459-464Crossref PubMed Scopus (80) Google Scholar) and (ii) phenolics (e.g. p-cresol, phenol) (5Stupperich E. Eisinger H.J. Kräutler B. Eur. J. Biochem. 1988; 172: 459-464Crossref PubMed Scopus (80) Google Scholar, 6Stupperich E. Eisinger H.J. Kräutler B. Eur. J. Biochem. 1989; 186: 657-661Crossref PubMed Scopus (37) Google Scholar). It is unclear why some cobamide-producing prokaryotes have evolved to synthesize these alternative cobamides. Cobamide-producing prokaryotes can be guided into synthesizing cobamides containing a lower ligand base different from the one usually found in the de novo synthesized cobamide by exogenously providing the alternative base to growing cells. Examples of this phenomenon termed “guided biosynthesis” have been documented (1Johnson M.G. Escalante-Semerena J.C. J. Biol. Chem. 1992; 267: 13302-13305Abstract Full Text PDF PubMed Google Scholar, 7Mervyn L. Smith E. Prog. Indust. Microbiol. 1964; 5: 152-201Google Scholar,8Stupperich E. Steiner I. Eisinger H.J. J. Bacteriol. 1987; 169: 3076-3081Crossref PubMed Google Scholar). Friedmann et al. (9Friedmann H.C. Cagen L.M. Annu. Rev. Microbiol. 1970; 24: 159-208Crossref PubMed Scopus (37) Google Scholar) proposed that the cobalamin biosynthetic enzyme nicotinate-mononucleotide (NaMN):Me2Bza phosphoribosyltransferase plays an important role in the incorporation of alternative lower ligand bases into cobamides in nature and during guided biosynthesis. This proposal was based on the observation that partially purified NaMN:Me2Bza phosphoribosyltransferases from Propionibacterium shermanni and from Clostridium sticklandii were both shown to use a variety of base substrates in place of Me2Bza in vitro (10Friedmann H.C. J. Biol. Chem. 1965; 240: 413-418Abstract Full Text PDF PubMed Google Scholar, 11Fyfe J.A. Friedmann H.C. J. Biol. Chem. 1969; 244: 1659-1666Abstract Full Text PDF PubMed Google Scholar). It was proposed that this lack of base substrate specificity was likely responsible for the incorporation of alternative bases into cobamidesin vivo. However, the incorporation of alternative NaMN:Me2Bza phosphoribosyltransferase products generatedin vitro into active cobamides in vivo was not demonstrated. In this paper we report the purification and biochemical characterization of the NaMN:Me2Bza phosphoribosyltransferase (CobT) from S. typhimurium. Previously we established a correlation between the presence of CobT and phosphoribosyltransferase activity using crude cell-free extracts containing increased levels of CobT protein (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). In this paper we demonstrate that homogeneous CobT is necessary and sufficient for the synthesis ofN 1-(5-phospho-α-D-ribosyl)-5,6-dimethylbenzimidazole (α-ribazole 5′-phosphate) (Fig. 2) and that the enzyme phosphoribosylates a wide range of alternative base substrates in vitro. We also present evidence that the resulting ribotides are incorporated into active cobamides in vivo. These results strongly support that CobT is responsible for the incorporation of alternative lower ligands into cobamides in S. typhimurium. Additionally, this result may help explain the inability to isolate mutants blocked in the synthesis of Me2Bza in this bacterium. The two bacterial strains used in this work were derivatives ofS. typhimurium strain LT2 and contained metE205 ara-9 mutations in the background. Strain JE2461 (cobA367::Tn10d(Tc)/pGP1–2 T7rpo + kan + pJO27 bla + + cobT + (cloning vector: pT7–5)) (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar) was used to increase the cellular level of CobT. Strain JE3762 (metE205 ara-9 cobB1206::MudJ DELcob291 strA1) was used in the biological activity assay system described below. DELcob291 spans the entire cobT gene (13Escalante-Semerena J.C. Johnson M.G. Roth J.R. J. Bacteriol. 1992; 174: 24-29Crossref PubMed Google Scholar). The composition of complex and chemically defined culture media and the concentration of nutritional supplements and antibiotics have been reported (14Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar, 15Davis R.W. Botstein D. Roth J.R. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 201-207Google Scholar). Growth conditions for cobT overexpression were as follows. One-liter cultures of strain JE2461 were grown with shaking (180 orbits/min) in Luria-Bertani medium supplemented with ampicillin (60 μg/ml) and kanamycin (60 μg/ml) in 2-liter Erlenmeyer flasks at 30 °C for 3.5 h (about 3 × 108 colony-forming units/ml; 55–70 Klett units). Overexpression of the CobT protein was induced by transferring the cultures to a 42 °C shaking water bath (200 rpm). After 30 min at 42 °C, the cultures were transferred to 37 °C and incubated with shaking (180 orbits/min) for 3 h. Growth conditions for the biological activity assays were as follows. Cells grown overnight in complex medium were pelleted and washed three times with 14.5 mm sterile NaCl. Approximately 108 cells were resuspended in 3 ml of molten 0.7% (w/v) agar and overlaid on one of the following: (i) Vogel-Bonner minimal medium containing glucose (11 mm) as carbon and energy source and cobinamide (20 nm); or (ii) No carbon Vogel-Bonner minimal medium containing 1,2-propanediol (50 mm) or ethanolamine (20 mm) as carbon and energy source, cobinamide (20 nm), and methionine (0.3 mm). Cbl (7 pmol) was spotted onto each plate to serve as the positive control; Me2Bza, 4,5-dimethyl-1,2-phenylenediamine (Me2Pda), Bza, histidine, adenine, guanine, or imidazole (150 nmol) was spotted and served as the negative control. Plates were incubated at 37 °C. Cbl biosynthesis was assessed in vivo by demanding strain JE3762 to grow under conditions that required the synthesis and utilization of Cbl. Strain JE3762 was unable to synthesize Cbl from its precursors cobinamide and Me2Bza but synthesized Cbl when provided with α-ribazole 5′-phosphate. Two in vivobiological activity assays with different sensitivities were employed. Methionine synthase catalyzes the last step in methionine biosynthesis. The metEand metH genes of S. typhimurium LT2 encode different methionine synthase enzymes. The activity of MetH (EC2.1.1.13) is Cbl-dependent, whereas that of MetE (E.C.2.1.1.14) is Cbl-independent (16Taylor R.T. Weissbach H. Boyer P.D. The Enzymes. 9. Academic Press, New York1973: 121-165Google Scholar). Strain JE3762 carries ametE mutation that forces the cells to synthesize methionine using the MetH enzyme. Therefore, derivatives of metEmutants unable to synthesize Cbl are phenotypically methionine or Cbl auxotrophs (17Jeter R.M. Olivera B.M. Roth J.R. J. Bacteriol. 1984; 159: 206-213Crossref PubMed Google Scholar). Approximately 25 Cbl molecules/cell satisfy the methionine requirement of a growing cell (18Bradbeer C. Dolphin D. B12. 2. John Wiley and Sons, New York1982: 31-56Google Scholar). Ethanolamine ammonia-lyase (EC 4.3.1.7), the first enzyme in ethanolamine catabolism in S. typhimuriumLT2, requires Ado-Cbl to function (19Chang G.W. Chang J.T. Nature. 1975; 254: 150-151Crossref PubMed Scopus (61) Google Scholar). 1,2-Propanediol dehydratase (EC4.2.1.28), which catalyzes the first step in 1,2-propanediol catabolism in this bacterium, is also an Ado-Cbl-dependent enzyme (20Poznanskaja A.A. Tanizawa K. Soda K. Toraya T. Fukui S. Arch. Biochem. Biophys. 1979; 194: 379-386Crossref PubMed Scopus (45) Google Scholar,21Jeter R.M. J. Gen. Microbiol. 1990; 136: 887-896Crossref PubMed Scopus (72) Google Scholar). Therefore, strains that cannot synthesize Ado-Cbl are unable to utilize 1,2-propanediol or ethanolamine as carbon and energy sources. Approximately 500 Cbl molecules/cell are needed for continued growth on ethanolamine (18Bradbeer C. Dolphin D. B12. 2. John Wiley and Sons, New York1982: 31-56Google Scholar). The in vitro phosphoribosyltransferase assay relies on changes in the chromatographic behavior of radiolabeled Me2Bza as described elsewhere (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). CobT protein was diluted with glycine-NaOH buffer (50 mm, pH containing and mm). The concentration of in the was The was and contained homogeneous CobT and glycine-NaOH buffer pH were for 10 min at 37 °C A of CobT activity was defined as the of enzyme needed to synthesize of α-ribazole This assay was used for different as described below. The contained Me2Bza (20 and (20 The of enzyme was within the of the The of protein used for each purification step crude cell-free CobT CobT were in and The contained Me2Bza (20 and (20 were at pH each the of is optimal pH the were pH pH pH pH CHES, pH pH pH and MOPS, pH The product of the was using a After of product 5′-phosphate) from by the was to a for and the of in an assay to was was from a control. The of in a (7 from the was assessed by to the of in the The of in the assay to product was Me2Bza was synthesized as described (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar) with the of and of were used in a was in at °C. The for were by at a concentration nmol) the concentration was the analysis of Me2Bza, a concentration of nmol) was used the Me2Bza concentration was from were with the analysis mononucleotide was for in containing of and a concentration of from three were with the analysis and nmol) was for in Bza, Me2Pda, imidazole, histidine, adenine, or guanine was to the in of these compounds were not of products was using a biological activity assay the the of strain JE3762 to a active growth was increase the of assays were to ml and contained of CobT and 3 of were at pH incubated for h at 37 °C, at °C for for 10 min × in a and the under with a were resuspended in of and a was for biological assays were CobT to demonstrate the of the biological activity on CobT. were using in place of The used of and of CobT. The assay was incubated at 37 °C for purification of the CobT product was as described (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). mass analysis of CobT was using a × with mm pH at a of was at used and A was were and The for CobT was analysis of the CobT products was as described (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar) with the gel (20 × 10 were by were on the were for approximately 45 min using and fast atom bombardment mass of the CobT product were as described (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). purification were at °C. The buffer used the purification was mm pH to this buffer are below. The used the purification to the contained or as to the A 2-liter culture of strain JE2461 was grown under conditions that overexpressed The resulting of was resuspended in 45 ml of After the of cells were by using a of protein during the was at a °C. was by at × h was to the to incubated at °C for 30 and for min at × to was to the The was onto a × a with buffer containing m and The was and at a of 30 After the was washed with 30 ml of the buffer by a that the of from to and increased the concentration from to the was by a with buffer containing m CobT from the the of the containing CobT were by and CobT were and onto the The was onto a × with buffer containing m at a of 25 After the the was washed with ml of buffer containing m and mm the was with a to buffer containing m and the was washed with ml of buffer containing m and m CobT within the with the of protein at mm NaCl. A of the homogeneous CobT as from These homogeneous were using a and overnight buffer containing The enzyme was and at °C in a The NH2-terminal sequence of purified CobT was at the at the of using sequence was with a were reported using the system were by a of the reported by J. Gen. PubMed Scopus Google Scholar) using a using of These to using the which is based on the Biochem. PubMed Scopus Google Scholar). was with polyacrylamide Nature. 1970; PubMed Scopus Google Scholar) and with J. Smith J.A. K. in Wiley New Scholar). and The of CobT was as reported D.M. John Wiley and Sons, New Scholar). were using a were with a cobT was overexpressed using a was from a S. Smith J.A. K. in 2. Wiley New Scholar). The of this was described G.A. Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar). on of CobT was to be approximately of the protein in crude cell-free of the strain not The of CobT in this system was G.A. Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar). The purification of CobT from crude cell-free is in I. CobT activity in the crude was h and at °C. This was lower than the activity or of CobT. the of the activity not of continued at °C not for these results may be that was responsible for or CobT also or purification in a The purification of CobT is shown in CobT was 97% as by of The NH2-terminal sequence of homogeneous CobT was by The sequence of homogeneous CobT was to that from the sequence that the cobT product been purified. Additionally, the sequence established that as elsewhere J. Roth J.R. J. Bacteriol. PubMed Scopus Google the was 30 bases of the proposed J.R. S. J. Bacteriol. PubMed Google Scholar). using crude cell-free containing increased levels of we that CobT was the NaMN:Me2Bza phosphoribosyltransferase responsible for the synthesis of α-ribazole an in the of the of Cbl (Fig. 2) (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). homogeneous we these to the for this activity in vitro. After the identity of the product was confirmed by and mass analysis to be α-ribazole not In the product was to be the of strain to synthesize Cbl. Therefore, was that homogeneous CobT was necessary and sufficient to α-ribazole from and vitro. The optimal for CobT activity was 45 °C CobT of its activity at °C and at 30 °C activity was at 45 °C, activity assays were at the of 37 °C. The optimal pH for CobT activity was pH CobT of its activity at pH and at pH CobT was for 10 min at by or with (20 or with Me2Bza (20 CobT was for phosphoribosyltransferase activity at 37 °C. The were with the one when CobT was on at 37 °C with in a in activity of the activity of CobT on However, CobT activity was to as high as °C when with We that a on CobT at increased Me2Bza not increase CobT at not CobT activity was at increased Me2Bza CobT activity at mm Me2Bza was with at mm CobT activity was with to protein concentration to CobT activity was with to to on the CobT was to be in with a mass of This to the of homogeneous CobT on (Fig. mass was by gel to be that CobT was a The two on to a of and from the for The of CobT at was to be using the as described L. PubMed Scopus Google Scholar). on from the apparentK m of CobT for was 680 was of and was of of CobT The apparent K m was to the CobT from C. sticklandii J.A. Friedmann H.C. J. Biol. Chem. 1969; 244: 1659-1666Abstract Full Text PDF PubMed Google Scholar) and than the B. D. L. D. L. J. J. Bacteriol. PubMed Google Scholar). The apparent K m of CobT for Me2Bza not be of the activity of However, we were to that the apparent K m was less than 10 not The a K m for Me2Bza of B. D. L. D. L. J. J. Bacteriol. PubMed Google Scholar). The K m for Me2Bza of the C. sticklandii was not and ribose were both in of in phosphoribosyltransferase CobT used as substrate in CobT phosphoribosyltransferase activity was at of this were from a that not on from three the apparent K m of CobT for was 30 mm, than the one for This the that is the in vivo substrate for CobT. The was of and the was of of CobT The CobT utilization of was in to the C. sticklandii which not utilize as a substrate J.A. Friedmann H.C. J. Biol. Chem. 1969; 244: 1659-1666Abstract Full Text PDF PubMed Google Scholar). was not utilized by CobT. activity assays were with a variety of bases in place of Me2Bza including Bza, Me2Pda, imidazole, histidine, adenine, and of product was using biological activity the the of strain JE3762 to an active growth was No was to the growth results are shown in and containing Me2Bza, Me2Pda, or in a product that growth when methionine synthase 1,2-propanediol dehydratase not and ethanolamine ammonia-lyase not However, imidazole, histidine, adenine, and guanine growth when ethanolamine ammonia-lyase (Fig. and 1,2-propanediol dehydratase not but not methionine synthase not These demonstrate CobT of a variety of base of cobamides from the CobT products in and utilization of the resulting cobamides by in vivo. The of these results are under of CobT utilization of histidine, adenine, guanine, and imidazole using the ethanolamine ammonia-lyase biological activity CobT phosphoribosyltransferase were with guanine and imidazole in place of Growth of strain JE3762 in to the CobT that CobT utilized histidine, adenine, guanine, and imidazole in that the products were into active cobamides utilized by ethanolamine ammonia-lyase in vivo. activity assays were as described under CobT to containing enzyme. is the No CobT was CobT utilization of was by phosphoribosyltransferase activity assays using in place of by of product from by the ribose been reported to be J.A. Friedmann H.C. J. Biol. Chem. 1969; 244: 1659-1666Abstract Full Text PDF PubMed Google the assays were with or of the of assay CobT utilized mm) as a substrate with a activity of less active than the positive Me2Bza We have CobT of a variety of bases as substrates in vitro and the incorporation of these alternative products into active cobamides in vivo. We that the resulting cobamides in the of the histidine, adenine, guanine, and imidazole are not into Cbl growth was when methionine synthase activity was This that S. typhimurium incorporated the CobT in vitro products into a vivo. These results support for the role of the NaMN:Me2Bza phosphoribosyltransferase in the incorporation of alternative lower ligands into cobamides in nature and during guided biosynthesis. Additionally, these results demonstrate that methionine ethanolamine and 1,2-propanediol dehydratase have different for the identity of the lower Methionine synthase was than ethanolamine ammonia-lyase or 1,2-propanediol dehydratase in its utilization of different cobamides in vivo. is that the alternative lower ligands may the enzyme from the The of the methionine synthase that Cbl been S. J.T. 1994; PubMed Scopus Google Scholar). The that Me2Bza and the from the and are by the enzyme in a this the between Me2Bza and is by a between a of the enzyme and the lower ligands of the alternative cobamides in not into the or were into a the inability to was the for the lack of we that ethanolamine ammonia-lyase and 1,2-propanediol dehydratase cobamides than methionine The of the Cbl of ethanolamine ammonia-lyase and 1,2-propanediol dehydratase have not been to However, results that methionine synthase can use alternative cobamides that lower ligands to Me2Bza, the products of the and in the in of cobamides utilized by this enzyme. We cannot that may have been to vivo. However, in the of Bza, we that S. typhimurium a active cobamide with as the lower ligand when with (1Johnson M.G. Escalante-Semerena J.C. J. Biol. Chem. 1992; 267: 13302-13305Abstract Full Text PDF PubMed Google the that was in this This that methionine synthase is to utilize cobamide containing as the lower The of methionine synthase to utilize alternative cobamides is by et al. H. H. E. B. Biol. Chem. 1988; PubMed Scopus Google Scholar) that a variety of cobamides containing different lower ligands growth of an strain that required active cobamide for methionine synthase However, in to also growth with It been reported that some prokaryotes when with cobamides can the alternative lower ligands with base E. Steiner I. Eisinger H.J. J. Bacteriol. 1987; 169: 3076-3081Crossref PubMed Google Scholar, H.C. Cagen L.M. Annu. Rev. Microbiol. 1970; 24: 159-208Crossref PubMed Scopus (37) Google Scholar). this the in for and Smith L. Smith E. Prog. Indust. Microbiol. 1964; 5: 152-201Google Scholar) also of cobamides Cbl which support in synthase of Me2Bza biosynthetic genes and of products have been The Me2Bza auxotrophs isolated in S. typhimurium mutations We have proposed that are Me2Bza auxotrophs a protein to as for CobT activity when Me2Bza levels are increased (12Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). we have not that CobT may in to its phosphoribosyltransferase activity be in Me2Bza we that CobT is not responsible as elsewhere Roth J.R. J. Bacteriol. PubMed Google Scholar). are a of that may explain the with mutations in Me2Bza biosynthetic (i) of Me2Bza biosynthetic genes may be or (ii) than one Me2Bza biosynthetic in S. typhimurium. based on the results is that in vivo than one base can for Me2Bza not be the synthesis of lower ligands was We from the of the at the of for the mass