The PduX Enzyme of Salmonella enterica Is an l-Threonine Kinase Used for Coenzyme B12 Synthesis

Here, the PduX enzyme of Salmonella enterica is shown to be an l-threonine kinase used for the de novo synthesis of coenzyme B12 and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His6) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. 31P NMR spectroscopy established that purified PduX-His6 catalyzed the conversion of l-threonine and ATP to l-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: Vmax = 62.1 ± 3.6 nmol min–1 mg of protein–1; Km, ATP = 54.7 ± 5.7 μm and Km,Thr = 146.1 ± 8.4 μm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B12 de novo and from Cby, but not from cobinamide, which was the expected phenotype for an l-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with l-threonine-O-3-phosphate, providing further support that PduX is an l-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B12 as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an l-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free l-threonine and the first l-threonine kinase shown to function in coenzyme B12 synthesis. Here, the PduX enzyme of Salmonella enterica is shown to be an l-threonine kinase used for the de novo synthesis of coenzyme B12 and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His6) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. 31P NMR spectroscopy established that purified PduX-His6 catalyzed the conversion of l-threonine and ATP to l-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: Vmax = 62.1 ± 3.6 nmol min–1 mg of protein–1; Km, ATP = 54.7 ± 5.7 μm and Km,Thr = 146.1 ± 8.4 μm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B12 de novo and from Cby, but not from cobinamide, which was the expected phenotype for an l-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with l-threonine-O-3-phosphate, providing further support that PduX is an l-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B12 as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an l-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free l-threonine and the first l-threonine kinase shown to function in coenzyme B12 synthesis. The B12 coenzymes (adenosylcobalamin, AdoCbl, and methylcobalamin, MeCbl) are required cofactors for at least 15 different enzymes that are widely distributed in nature and are essential for human health (1Banerjee R. Chemistry and Biochemistry of B12. John Wiley and Sons, New York1999Google Scholar, 2Schneider Z. Stroinski A. Comprehensive B12: Chemistry, Biochemistry, Nutrition, Ecology, Medicine. Walter De Gruyter, Berlin1987Crossref Google Scholar). AdoCbl and MeCbl are synthesized de novo by certain prokaryotes and from corrinoid precursors by a broader range of organisms (1Banerjee R. Chemistry and Biochemistry of B12. John Wiley and Sons, New York1999Google Scholar, 2Schneider Z. Stroinski A. Comprehensive B12: Chemistry, Biochemistry, Nutrition, Ecology, Medicine. Walter De Gruyter, Berlin1987Crossref Google Scholar). The synthesis of B12 has been studied extensively in Salmonella enterica (3Escalante-Semerena J.C. J. Bacteriol. 2007; 189: 4555-4560Crossref PubMed Scopus (73) Google Scholar, 4Roth J.R. Lawrence J.G. Rubenfield M. Kieffer-Higgins S. Church G.M. J. Bacteriol. 1993; 175: 3303-3316Crossref PubMed Google Scholar). This organism carries out de novo synthesis under anaerobic conditions and assimilates corrinoids such as vitamin B12, cobinamide (Cbi), 2The abbreviations used are: Cbi, cobinamide; Cby, cobyric acid; AP-P, (R)-1-amino-2-propanol-O-2-phosphate; 1,2-PD, 1,2-propanediol; HPLC, high pressure liquid chromatography; AP, (R)-1-amino-2-propanol. and cobyric acid (Cby) under both aerobic and anaerobic conditions (3Escalante-Semerena J.C. J. Bacteriol. 2007; 189: 4555-4560Crossref PubMed Scopus (73) Google Scholar, 5Roth J.R. Lawrence J.G. Bobik T.A. Annu. Rev. Microbiol. 1996; 50: 137-181Crossref PubMed Scopus (434) Google Scholar). Corrinoid assimilation begins with transport into the cytoplasm by the B12 uptake system (btu) followed by synthetic steps specific to a particular corrinoid (Fig. 1) (6de Veaux L.C. Clevenson D.S. Bradbeer C. Kadner R.J. J. Bacteriol. 1986; 167: 920-927Crossref PubMed Google Scholar, 7Heller K. Kadner R.J. J. Bacteriol. 1985; 161: 904-908Crossref PubMed Google Scholar, 8Van Bibber M. Bradbeer C. Clark N. Roth J.R. J. Bacteriol. 1999; 181: 5539-5541Crossref PubMed Google Scholar). In the case of Cbi, an adenosyl group is added to the central cobalt atom to form adenosyl-Cbi, which is phosphorylated to adenosyl-Cbi-phosphate (9Escalante-Semerena J.C. Suh S.J. Roth J.R. J. Bacteriol. 1990; 172: 273-280Crossref PubMed Google Scholar, 10O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1995; 270: 23560-23569Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Subsequently, the nucleotide loop is assembled in three steps to form AdoCbl (3Escalante-Semerena J.C. J. Bacteriol. 2007; 189: 4555-4560Crossref PubMed Scopus (73) Google Scholar). Last, a portion of the AdoCbl is converted to MeCbl (11Fujii K. Huennekens F.M. J. Biol. Chem. 1974; 249: 6745-6753Abstract Full Text PDF PubMed Google Scholar, 12Matthews, R. G. (1999) in Chemistry and Biochemistry of B12 (Banerjee, R., ed) pp. 681–706, John Wiley & Sons, Inc., New YorkGoogle Scholar). Many of the steps used for Cbi assimilation are also used for the assimilation of Cby as well as the de novo synthesis of AdoCbl and MeCbl (Fig. 1). A key difference is that Cby assimilation and de novo synthesis require (R)-1-amino-2-propanol-O-2-phosphate (AP-P), whereas the conversion of Cbi to the B12 coenzymes does not (3Escalante-Semerena J.C. J. Bacteriol. 2007; 189: 4555-4560Crossref PubMed Scopus (73) Google Scholar) (Fig. 1). Prior studies showed that AP-P was produced by decarboxylation of l-threonine-O-3-phosphate (l-Thr-P), which was catalyzed by the CobD enzyme (13Brushaber K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-3691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). These findings predicted that an l-threonine (l-Thr) kinase would be required for the de novo synthesis of AdoCbl and MeCbl, and the assimilation of Cby. However, this enzyme has not been identified in any system. In S. enterica, AdoCbl and MeCbl are required cofactors for three enzymes (5Roth J.R. Lawrence J.G. Bobik T.A. Annu. Rev. Microbiol. 1996; 50: 137-181Crossref PubMed Scopus (434) Google Scholar). MeCbl-dependent methionine synthase is used to convert homocysteine to methionine (14Cauthen S.E. Foster M.A. Woods D.D. Biochem. J. 1966; 98: 630-635Crossref PubMed Scopus (33) Google Scholar); AdoCbl-dependent diol dehydratase and ethanolamine ammonia lyase are required for growth on 1,2-propanediol (1,2-PD) and ethanolamine, respectively (15Jeter R.M. J. Gen. Microbiol. 1990; 136: 887-896Crossref PubMed Scopus (72) Google Scholar, 16Roof D.M. Roth J.R. J. Bacteriol. 1988; 170: 3855-3863Crossref PubMed Scopus (117) Google Scholar). The genes specific for 1,2-PD utilization (pdu) are found in a large contiguous cluster (15Jeter R.M. J. Gen. Microbiol. 1990; 136: 887-896Crossref PubMed Scopus (72) Google Scholar, 17Bobik T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar). Bioinformatic studies tentatively suggest that the last gene of the pdu operon (pduX) encodes an l-Thr kinase (18Rodionov D.A. Vitreschak A.G. Mironov A.A. Gelfand M.S. J. Biol. Chem. 2003; 278: 41148-41159Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). The PduX protein has homology to the GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family of kinases and a number of PduX homologues are encoded by genes located proximal to genes for AdoCbl biosynthesis. However, no experimental studies of PduX homologues have been conducted in any system. Here, we present genetic and biochemical studies that show PduX is an l-Thr kinase used for the de novo synthesis of AdoCbl and the assimilation of Cby. To our knowledge, PduX is the first enzyme shown to phosphorylate free l-Thr. Chemicals and Reagents—Antibiotics, Cbi, l-Thr-P, (R)-1-amino-2-propanol (AP), nucleoside triphosphates, and nucleoside diphosphates were purchased from Sigma. Isopropyl β-d-thiogalactopyranoside was from Diagnostic Chemical Ltd., Charelottetown, Prince Edward Island, Canada. Restriction enzymes and T4 DNA ligase were from New England Biolabs, Beverly, MA. Pefabloc SC PLUS was purchased from ICN Biomedicals, Inc., Aurora, OH. Other chemicals were from Fisher Scientific. Cby was kindly provided by Kathy Krasny from the laboratory of J. Escalante-Semerena. Bacterial Strains, Media, and Growth Conditions—The bacterial strains used in this study are listed in Table 1. The minimal medium used was NCE supplemented with 0.4% 1,2-PD, 1 mm MgSO4, 50 μm ferric citrate, 1 μm 5,6-dimethylbenzimidazole, and 3 mm (each) valine, isoleucine, leucine, and threonine. LB (Luria-Bertani) medium was the rich medium used.TABLE 1Bacterial strains used in this and study enterica study study study study study in a was as J. A Scholar). DNA was purified by to the and DNA was purified by Restriction were out J. A Scholar). of DNA T4 DNA ligase was used to the was out as T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar). was by and to the was used to The protein of was by protein of for of PduX and was used to the pduX from T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar). The used for were and The used for to the of PduX was These and that were used for into or Havemann G.D. Bobik T.A. J. Bacteriol. PubMed Scopus Google Scholar). were into Escherichia by and were by on LB supplemented with or were from The of DNA was by or and the DNA of pduX was with the expected DNA were used for further DNA was out by the DNA of of from Growth are in the To the LB were at and were by and in growth were to a of and the growth was by the at a and well was with of and at as Havemann G.D. Bobik T.A. J. Bacteriol. 2007; 189: PubMed Scopus Google Scholar). Corrinoid and strains of S. enterica were on of LB medium supplemented with 1,2-PD, 1 μm 5,6-dimethylbenzimidazole, and μm at in were by and in 1 of 50 mm The was into The were and with for 3 The was for at on for and at for 1 at The corrinoids present in the were a bioassay on the AdoCbl-dependent growth of S. enterica on the ethanolamine minimal Growth were out a as for was on a of vitamin B12 of The was from to vitamin B12 with an of Growth of pduX strains used for expression of pduX were on of LB supplemented with at in a New with at were to an of at and protein expression was by the of mm were at with at for an and by at for at a of of S. of was in of 50 mm mm mm and by at Pefabloc SC PLUS was added to the to a of to The was at for at a to the and The was the used for enzyme of of from an expression was a of acid with 50 mm mm mm The was with of 50 mm mm mm the enzyme to the was with of 50 mm mm mm The purified protein was by and with of mm following the PduX Enzyme of PduX was by an to the The a enzyme system to from with to form by kinase, and with in the of the 15 mm mm mm in a of was by the with a The was and was To were used kinetic the of certain as in the was on with GTP, CTP, or UTP. 31P NMR of the PduX were by 31P were conditions for the PduX enzyme with 1 mg of purified PduX-His6 in a of 1 The were at was by a were to NMR 50 of was added 31P NMR of and kinase were with a and a at the following and were with from Chemical were to which was to was used with a system that a a and a A and mm and 50 mm and 1 The was 1 and were with a from 50 to mm The for GTP, CTP, and were and PduX for S. enterica Growth on 1,2-PD with Cby, but on with kinase is predicted to be required for the synthesis of AdoCbl from Cby, but not from Cbi (Fig. 1). we aerobic growth of S. enterica and a pduX mutant on 1,2-PD minimal medium supplemented with Cby or growth the synthesis of AdoCbl from Cby or Cbi (15Jeter R.M. J. Gen. Microbiol. 1990; 136: 887-896Crossref PubMed Scopus (72) Google Scholar). showed that a pduX mutant was impaired for growth on 1,2-PD minimal medium supplemented with Cby, but well on medium supplemented with Cbi (Fig. In S. enterica well on 1,2-PD medium supplemented with Cby or The of S. enterica with Cbi and Cby were and whereas of the pduX mutant were and These are the expected of an l-Thr kinase mutant. growth with Cbi that the 1,2-PD is and that corrinoid uptake is a system is used for uptake of Cbi, Cby, and corrinoids (6de Veaux L.C. Clevenson D.S. Bradbeer C. Kadner R.J. J. Bacteriol. 1986; 167: 920-927Crossref PubMed Google Scholar, 7Heller K. Kadner R.J. J. Bacteriol. 1985; 161: 904-908Crossref PubMed Google Scholar, 8Van Bibber M. Bradbeer C. Clark N. Roth J.R. J. Bacteriol. 1999; 181: 5539-5541Crossref PubMed Google Scholar). also that pduX mutants out the steps for the conversion of Cbi to AdoCbl (Fig. 1). the of pduX mutants to Cby be to a defect in the conversion of Cby to (Fig. 1). This 1) an an enzyme that to an and an l-Thr kinase (9Escalante-Semerena J.C. Suh S.J. Roth J.R. J. Bacteriol. 1990; 172: 273-280Crossref PubMed Google Scholar, K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-3691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Escalante-Semerena J.C. J. Bacteriol. 2007; 189: PubMed Scopus Google Scholar). The and CobD enzymes have been in S. enterica (9Escalante-Semerena J.C. Suh S.J. Roth J.R. J. Bacteriol. 1990; 172: 273-280Crossref PubMed Google Scholar, K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-3691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Escalante-Semerena J.C. J. Bacteriol. 2007; 189: PubMed Scopus Google Scholar). the studies suggest that PduX is an l-Thr kinase used for the conversion of Cby to A pduX by of pduX expression was into a pduX mutant by This corrected the growth defect of a pduX mutant on 1,2-PD minimal medium supplemented with Cby was compared with for (Fig. In on growth is This showed that the phenotype growth with from the pduX but not from or an of Growth with the Growth of a pduX mutants are corrected by supplementation of growth medium with a conducted here showed that μm corrected the growth defect of a pduX mutant on 1,2-PD Cby (Fig. μm the for a pduX mutant was compared with a of for the strains a at of These that PduX has a in the synthesis of for of a pduX studies showed that a mutant was corrected for the synthesis of MeCbl from Cby by of to growth C. Roth J.R. J. Bacteriol. PubMed Google Scholar). This and findings that an kinase phosphorylated to AP-P the defect (Fig. 1) (13Brushaber K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-3691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). PduX was for kinase However, were a mutant and a pduX mutant on minimal medium supplemented with Cby and that S. enterica to AP-P in the of PduX not of 1 and mm were and in no case the the The studies were conducted in a under conditions the conversion of Cby to MeCbl is required for growth in minimal medium R.M. Roth J.R. J. Bacteriol. PubMed Google Scholar). of PduX for kinase 1,2-PD minimal medium supplemented with Cby and enzyme assays with purified enzyme were also not that PduX does not phosphorylate PduX for S. enterica Growth on with Cby, but on with pduX gene is the last gene in the pdu operon T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar). expression of this operon 1,2-PD (15Jeter R.M. J. Gen. Microbiol. 1990; 136: 887-896Crossref PubMed Scopus (72) Google Scholar, T.A. M. Roth J.R. J. Bacteriol. PubMed Google Scholar, Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar, M. Bobik T.A. Roth J.R. J. Bacteriol. 1993; 175: PubMed Google Scholar). This the of PduX ethanolamine showed that a pduX mutant was to on ethanolamine minimal medium supplemented with Cby, but well on medium supplemented with Cbi (Fig. In S. enterica well on ethanolamine medium supplemented with Cby or The of S. enterica with Cbi and Cby were and whereas of the pduX mutant were and In addition, the growth defect of the pduX mutant on ethanolamine with Cby was corrected by ectopic expression of this defect from the pduX but not from or an not pduX is used for AdoCbl synthesis in the of 1,2-PD, which is required for of the pdu operon (15Jeter R.M. J. Gen. Microbiol. 1990; 136: 887-896Crossref PubMed Scopus (72) Google Scholar, 17Bobik T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar, Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar, M. Bobik T.A. Roth J.R. J. Bacteriol. 1993; 175: PubMed Google Scholar). This is to that pduX has a different genes to be in the but Growth of a pduX on with growth defect of a pduX mutant on minimal ethanolamine Cby was corrected by of but not by of AP-P not This was to with 1,2-PD and provided further that PduX is an l-Thr kinase. of MeCbl to of enterica mutants require MeCbl for methionine l-Thr kinase be required for growth of strains on minimal medium supplemented with Cby (Fig. 1) (14Cauthen S.E. Foster M.A. Woods D.D. Biochem. J. 1966; 98: 630-635Crossref PubMed Scopus (33) Google Scholar). However, showed that a pduX mutant on minimal medium supplemented with Cby. for the and pduX mutant were and This that PduX to MeCbl but S. enterica also produced a l-Thr kinase that growth of a mutant as is further under PduX for the de of kinase is expected to be required for the de novo synthesis of AdoCbl (Fig. 1) (13Brushaber K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-3691Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). we used a bioassay to de novo synthesis of AdoCbl and MeCbl by S. enterica, a pduX mutant and a mutant These strains produced B12 in the following of ± ± and a pduX mutant produced as B12 as the S. enterica was used for the This on ethanolamine minimal medium supplemented with corrinoids such as AdoCbl and MeCbl, but not on medium supplemented with corrinoids such as which in a pduX mutant. of the PduX-His6 with a C-terminal tag (PduX-His6) was produced at high levels an expression system and purified by (Fig. This the of PduX-His6 that following The of PduX-His6 by was which was to the expected of and the of the PduX-His6 NMR spectroscopy was used to the of the PduX the 31P NMR of a kinase to of purified PduX-His6 The at to the of the at and to the and of the 31P NMR of the The at and to the and of the of at with of purified The to the of ATP is The at and of the well with the of the (Fig. The at to the group of (Fig. the of at with of purified that the l-Thr as well as both ATP and (Fig. that and are the of the PduX In of purified PduX-His6 l-Thr kinase we used a that the nucleoside under ATP as the the specific of purified PduX-His6 was nmol min–1 mg of In the of no was In the of or by The to have from enzyme assays that PduX has l-Thr kinase and of the PduX as well as an were used to determine the substrate of ATP to the of PduX-His6 with CTP, GTP, or was and by the and and by 3 μm purified PduX-His6 and μm nucleoside The of PduX on enzyme was also showed that the of was to PduX-His6 from to μm μm ATP and μm l-Thr were used as regression an of and Vmax for PduX-His6 displayed Michaelis-Menten kinetics with respect to both ATP and l-Thr (Fig. on nonlinear the for ATP and l-Thr were 54.7 ± 5.7 and 146.1 ± 8.4 The enzyme Vmax was ± 3.6 nmol min–1 mg of l-Thr was and ± 3.6 nmol min–1 mg of ATP was The of is 62.1 ± 3.6 nmol min–1 mg of kinetic were from The for ATP and l-Thr were ± and ± Vmax were ± nmol min–1 mg of l-Thr was and ± nmol min–1 mg of ATP was The of is ± nmol min–1 mg of the for ATP were levels of l-Thr were added to the of levels of ATP were added to assays the for l-Thr were PduX-His6 was used at a of 3 μm. The used for kinetic were the of three of the in a number of has steps in the de novo synthesis of AdoCbl and MeCbl (3Escalante-Semerena J.C. J. Bacteriol. 2007; 189: 4555-4560Crossref PubMed Scopus (73) Google Scholar, 5Roth J.R. Lawrence J.G. Bobik T.A. Annu. Rev. Microbiol. 1996; 50: 137-181Crossref PubMed Scopus (434) Google Scholar, PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, J. Bacteriol. PubMed Scopus Google Scholar, J. Chem. 2003; PubMed Scopus Google Scholar, Escalante-Semerena J.C. PubMed Scopus Google Scholar). particular to this are the studies by (13Brushaber K.R. O'Toole G.A. Escalante-Semerena J.C. J. Biol. Chem. 1998; 273: 2684-3691Abstract Full Text Full Text PDF PubMed Scopus (43) Google which showed the CobD enzyme the decarboxylation to This predicted that B12 synthesis would require an l-Thr kinase. Subsequently, (18Rodionov D.A. Vitreschak A.G. Mironov A.A. Gelfand M.S. J. Biol. Chem. 2003; 278: 41148-41159Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) that PduX homologues be l-Thr kinases in B12 synthesis. This was on to the GHMP family of kinases and the that pduX genes were proximal to B12 genes in However, is well that on and gene are In addition, a number of PduX homologues are encoded by genes to B12 genes the of PduX Here, we presented in and in that the pduX gene of S. enterica encodes an l-Thr kinase in the de novo synthesis AdoCbl and the assimilation of Cby. 31P NMR spectroscopy that purified PduX-His6 the conversion of ATP and l-Thr to and (Fig. Enzyme assays the ATP was the preferred substrate for PduX compared with GTP, CTP, and UTP. The Vmax of PduX nmol min–1 mg of is the range for B12 enzymes that have in with the levels of AdoCbl and MeCbl that are required to support (5Roth J.R. Lawrence J.G. Bobik T.A. Annu. Rev. Microbiol. 1996; 50: 137-181Crossref PubMed Scopus (434) Google Scholar). The of PduX for ATP is well the ATP in and in Escherichia and Salmonella and J. K. and for Scholar). The for l-Thr is to for coli, which is a of S. enterica N. K. M. A. M. K. A. K. S. S. N. M. K. K. N. K. M. 2007; PubMed Scopus Google Scholar). in studies that purified PduX has l-Thr kinase with kinetic to a in AdoCbl synthesis. In to the in genetic that PduX has a in the synthesis of AdoCbl in A bioassay and growth showed that a pduX mutant was impaired for the de novo synthesis of AdoCbl and for the assimilation of Cby. In addition, the growth of a pduX mutant were corrected by of to minimal that PduX is for synthesis. A of is that found B12 in under aerobic is well established that S. enterica B12 de novo in the of R.M. Roth J.R. J. Bacteriol. PubMed Google Scholar). The conducted here and studies in our laboratory that S. enterica B12 under aerobic conditions at high and A. is by B12 synthesis. This the that S. enterica B12 in the of but for of ethanolamine and 1,2-PD as and (5Roth J.R. Lawrence J.G. Bobik T.A. Annu. Rev. Microbiol. 1996; 50: 137-181Crossref PubMed Scopus (434) Google Scholar). The genetic here also that a pduX mutant synthesized MeCbl to support growth of a mutant. This that S. enterica an l-Thr kinase in to but the is this kinase to support ethanolamine or 1,2-PD and is that the kinase is growth on the of the kinase be for 1,2-PD and ethanolamine A pduX mutant on 1,2-PD minimal medium supplemented with Cby (Fig. a kinase with Prior studies showed that the MeCbl to support growth of a mutant was the of AdoCbl to support 1,2-PD and ethanolamine a l-Thr kinase that has compared with PduX is To our knowledge, PduX is the first enzyme to a group to free l-Thr. that PduX is a of the GHMP kinase family (18Rodionov D.A. Vitreschak A.G. Mironov A.A. Gelfand M.S. J. Biol. Chem. 2003; 278: 41148-41159Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). This family that the group of ATP to such as and protein C. A. 1993; PubMed Scopus Google Scholar). A of PduX we is is that free which is also required for protein synthesis. PduX would to be to l-Thr or a that However, of the of the PduX enzyme has not been J. C. from the of for providing the cobyric acid used in this from the for with NMR

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