CobB, a New Member of the SIR2 Family of Eucaryotic Regulatory Proteins, Is Required to Compensate for the Lack of Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase Activity in cobT Mutants during Cobalamin Biosynthesis in Salmonella typhimurium LT2

The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of ∼28 kDa. The cobB gene was defined genetically by deletions (10O'Toole G.A. Rondon M.R. Escalante-Semerena J.C. J. Bacteriol. 1993; 175: 3317-3326Crossref PubMed Google Scholar), insertions (5Escalante-Semerena J.C. Johnson M.G. Roth J.R. J. Bacteriol. 1992; 174: 24-29Crossref PubMed Google Scholar), and point mutations (15Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google Scholar). The precise location of a Tn10d(Tc) element withincobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3′ of the potABCD operon, with the potABCD operon and cobB being divergently transcribed. cobB was overexpressed to ∼30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediateN 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole (also known as α-ribazole-5′-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997)J. Biol. Chem. 272, 17662–17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR andYTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996)J. Bacteriol. 178, 7016–7019). The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of ∼28 kDa. The cobB gene was defined genetically by deletions (10O'Toole G.A. Rondon M.R. Escalante-Semerena J.C. J. Bacteriol. 1993; 175: 3317-3326Crossref PubMed Google Scholar), insertions (5Escalante-Semerena J.C. Johnson M.G. Roth J.R. J. Bacteriol. 1992; 174: 24-29Crossref PubMed Google Scholar), and point mutations (15Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google Scholar). The precise location of a Tn10d(Tc) element withincobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3′ of the potABCD operon, with the potABCD operon and cobB being divergently transcribed. cobB was overexpressed to ∼30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediateN 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole (also known as α-ribazole-5′-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997)J. Biol. Chem. 272, 17662–17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR andYTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996)J. Bacteriol. 178, 7016–7019). cobalamin nutrient broth MudI1734 Tn10DEL16DEL17 phosphoribosyltransferase 5,6-dimethylbenzimidazole synthesizesN 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole N 1-(α-d-ribosyl)-5,6-dimethylbenzimidazole dicyanocobinamide adenosylcobalamin cyanocobalamin propionate phosphoribosylpyrophosphate tetracycline tetracycline-resistant chloramphenicol chloramphenicol-resistant kanamycin kanamycin-resistant plaque-forming unit kilobase pair. The genetic analysis of the late steps of cobalamin (Cbl)1 biosynthesis (also known as nucleotide loop assembly) in Salmonella typhimurium(1Rondon M.R. Trzebiatowski J.R. Escalante-Semerena J.C. Prog. Nucleic Acids Res. 56: 347-384Crossref PubMed Scopus (30) Google Scholar) has raised important questions about the biochemical capabilities of the enzymes involved (2Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar, 3Chen P. Ailion M. Weyand N. Roth J. J. Bacteriol. 1995; 177: 1461-1469Crossref PubMed Google Scholar). The specific step of the nucleotide loop assembly pathway that we address in this paper is the activation of the lower ligand base 5,6-dimethylbenzimidazole (Me2Bza). This step is catalyzed by the CobT enzyme, which converts Me2Bza to its 5′-mononucleotide (also known as α-ribazole-5′-phosphate) by transferring the phosphoribosyl group from NaMN to Me2Bza (Fig. 1) (2Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar, 4Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1997; 272: 17662-17667Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). As predicted by the biochemistry of the CobT reaction, cobalamin biosynthesis in mutants lacking CobT can be restored by providing α-ribazole-5′-phosphate in the medium (4Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1997; 272: 17662-17667Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). What was unexpected, however, was the finding that all previously reported Me2Bza auxotrophs (5Escalante-Semerena J.C. Johnson M.G. Roth J.R. J. Bacteriol. 1992; 174: 24-29Crossref PubMed Google Scholar) were alleles of cobT (2Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar), including insertions that eliminated CobT completely from cell-free extracts (6Trzebiatowski J.R. BacteriologyPh.D. thesis. University of Wisconsin-Madison, Madison, WI1998Google Scholar). This phenotype was difficult to explain in light of the documented biochemical activity of CobT as the NaMN:Me2Bza phosphoribosyltransferase (PRTase) (EC 2.4.2.21) (4Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1997; 272: 17662-17667Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This finding basically said that increasing the substrate for CobT would circumvent the lack of this enzyme. To help explain this paradox, we postulated the existence of an alternative enzyme that could perform the CobT reaction, with the qualification that such an enzyme would have less affinity for Me2Bza than CobT to explain the requirement for additional Me2Bza (2Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar). In this paper we report the identification of the cobB gene whose product is required to compensate for the lack of NaMN:Me2Bza PRTase activity in cobT mutants.cobB has been defined genetically by mutation analysis and physically by its nucleotide sequence (GenBankTM accession number U89687). Computer analysis of the primary amino acid sequence of CobB shows it to be a member of the SIR2 family of eucaryotic regulatory proteins. A list of strains, plasmids, and their genotypes are presented in Table I. Culture media composition, concentrations of antibiotics, and nutritional supplements were as reported (2Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar, 5Escalante-Semerena J.C. Johnson M.G. Roth J.R. J. Bacteriol. 1992; 174: 24-29Crossref PubMed Google Scholar, 7Davis R.W. Botstein D. Roth J.R. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 201-208Google Scholar). Increase in cell density was monitored at 650 nm with a Spectronic 20D spectrophotometer (Milton Roy Co., Rochester, NY). Five-ml cultures were grown with shaking (∼200 rpm) in 18 × 150-mm Pyrex™ tubes. Doubling times were calculated graphically from semi-log plots of A 650 as a function of time (hours).Table IStrains and plasmidsStrainGenotypeSource/Ref.E. coli DH5αF′F′/endA! hsdR17 (rk− mk+) supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacZYA-argF) U169 deoR(f80dlacD (lacZ)M15)46Raleigh E.A. Lech K. Brent R. Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. in Current Protocols in Molecular Biology. 1. Wiley Interscience, New York1989: 1.4.1-1.4.14Google Scholar, 47Woodcock D.M. Crowther P.J. Doherty J. Jefferson S. De Cruz E. Noyer-Weidner M. Smith S.S. Michael M.Z. Graham M.W. Nucleic Acids Res. 1989; 17: 3469-3478Crossref PubMed Scopus (640) Google ScholarS. typhimurium LT2 TR65831-aFormerly SA2929.metE205 ara-9K. Sanderson via J. Roth TN2644phoP::Tn10d(Cm)C. MillerDerivatives of TR6583 JE1391cobT111::Tn10DEL16DEL171-bAbbreviated in the text as Tn10d(Tc).hisF9951::Mud1–81-cAbbreviated in the text as MudA. JE1392cobT112::Tn10d(Tc)hisF9954::Mud1–8 JE1857cobT109::MudI17341-dAbbreviated in the text as MudJ. JE2445cobB1176::Tn10d(Tc) JE2501cobT109::MudI1734cobB1176::Tn10d(Tc) JE2593cobT109::MudI1734 DELcobB1177 JE2600cobT109::MudI1734 DELcobB1184 JE2607cobB1176::Tn10d(Tc)cobT109::MudI1734 recA1 JE2761pepT7::Mud(Cm) JE2699cobB1176::Tn10d(Tc)pepT7::MudI1734 JE2845cobB1206::MudI1734 JE3231cobB1206::Mud1–8 JE3727cobT111::Tn10d(Tc)cobB1206::MudJ JE4341cobB1176::Tn10d(Tc)cobT109::MudI1734 recA1/pCOBB1 JE4342cobB1176::Tn10d(Tc)cobT109::MudI1734 recA1/pCOBB2 JE4343cobT109::MudJ DELcobB1184/pCOBB3 JE4344cobB1176::Tn10d(Tc)cobT109::MudI1734 recA1/pCOBB4 JE4345cobB1176::Tn10d(Tc)cobT109::MudI1734 recA1/pCOBB5 JE4349DEL299(hisG-cob)/pGP1–2 T7rpo + kan +/pCOBB6 JE4350DEL299(hisG-cob)/pGP1–2 T7rpo + kan +/pT7–7bla +Plasmids pT7–5, -6, -7Overexpression vectors, bla +35Tabor S. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. Wiley Interscience, New York1990: 16.2.1.-16.2.11Google Scholar pGP1–2T7 ts rpo + kan +35Tabor S. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. Wiley Interscience, New York1990: 16.2.1.-16.2.11Google Scholar pSU19Cloning vector,cat +25Martı́nez E. Bartolome B. de la Cruz F. Gene (Amst.). 1988; 68: 159-162Crossref PubMed Scopus (253) Google Scholar pCOBB1∼9-kb Sau3A fragment containing cobB + cloned into theBamHI site of tetA in plasmid pBR328 (bla + cat +) pCOBB2∼5.5-kb EcoRI fragment of pCOBB1 containing cobB + cloned into pSU19 (cat +) pCOBB3pCOBB2cobB1176::Tn10d(Tc) pCOBB4∼1.8-kb SalI fragment of pCOBB2 containingcobB + cloned into pSU19 (cat +) pCOBB5∼1.3-kb SalI-NdeIcobB + fragment of plasmid pCOBB4 cloned into pSU19 (cat +) pCOBB6∼1-kbNdeI-NruI cobB + fragment of pCOBB5 cloned into overexpression vector pT7–7 (bla +)Unless otherwise stated the strains were generated during the course of these studies.1-a Formerly SA2929.1-b Abbreviated in the text as Tn10d(Tc).1-c Abbreviated in the text as MudA.1-d Abbreviated in the text as MudJ. Open table in a new tab Unless otherwise stated the strains were generated during the course of these studies. All genetic crosses were performed with the high transducing mutant phage P22 HT105 int-201 (8Schmieger H. Mol. Gen. Genet. 1971; 100: 378-381Crossref Scopus (120) Google Scholar, 9Schmieger H. Bakhaus H. Mol. Gen. Genet. 1973; 120: 181-190Crossref PubMed Scopus (56) Google Scholar) as described elsewhere (7Davis R.W. Botstein D. Roth J.R. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 201-208Google Scholar). Crosses that selected for kanamycin-resistant (Kmr) or chloramphenicol-resistant (Cmr) transductants were first preincubated for at shaking (10O'Toole G.A. Rondon M.R. Escalante-Semerena J.C. J. Bacteriol. 1993; 175: 3317-3326Crossref PubMed Google Scholar). mutations in cobB were isolated by using as described elsewhere (7Davis R.W. Botstein D. Roth J.R. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 201-208Google Scholar, S. A. 1971; 68: PubMed Scopus Google Scholar). A P22 phage grown was for which an of the from × to × was as to to chloramphenicol medium containing and were medium D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) containing and dicyanocobinamide and 5,6-dimethylbenzimidazole or cobB mutants were identified by their to medium containing and Me2Bza and by their medium containing a nucleotide loop assembly The propionate phenotype of cobB mutants was as described elsewhere O'Toole G.A. Trzebiatowski J.R. D. Escalante-Semerena J.C. PubMed Google Scholar, Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar). in cobB were Tn10d(Tc) element in cobB was isolated from a of strains of which was to one Tn10d(Tc) element in the P22 phage grown this was as to to tetracycline medium containing and was identified as described for the point A was to MudI1734 (15Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google in this a phage grown a of strains a element in their was as to to kanamycin medium containing and to medium with and supplements was to strains isolated mutants and nucleotide loop were of cobB were isolated in by the of J. Bacteriol. PubMed Google Scholar) as by and J. Bacteriol. PubMed Google Scholar). A from an of grown in medium containing tetracycline was and at and kanamycin-resistant (Kmr) that the to medium containing and Me2Bza were to a of The of the deletions was The location of cobB in the was determined by using the of and J. Bacteriol. 1992; 174: PubMed Google Scholar, Genetics. 1989; Google Scholar). was grown in medium containing and J. Bacteriol. PubMed Google Scholar, J. Bacteriol. PubMed Google Scholar) to the of of P22 were from a of strains containing one or in a known site of the chromosome. were as reported P. P. Genetics. 1988; PubMed Google Scholar) and by means of a of A location of cobB was by and crosses with genetic within the region by the to crosses were performed using phage grown + as and as for transductants and and to the of of the All strains for crosses mutations and were from medium containing was in all crosses D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and were at concentrations described The direction of transcription of cobB was determined to be by the of and Roth Roth J.R. Genetics. PubMed Google Scholar) as described elsewhere J.C. Roth J.R. J. Bacteriol. PubMed Google Scholar). The of phage grown the strains was determined as described (7Davis R.W. Botstein D. Roth J.R. A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980: 201-208Google Scholar). The element was to as reported (15Castilho B.A. Olfson P. Casadaban M.J. J. Bacteriol. 1984; 158: 488-495Crossref PubMed Google Scholar) to the insertions and the of mutant cobB + +) was with phage grown and or at an of of 1. transductants were selected medium containing at and to medium containing as and A plasmid the of cobB was from a of Sau3A by of the S. The Sau3A were cloned into the site of the tetA gene of plasmid pBR328 by C. University of This plasmid is to as and it is shown in 2. P22 grown the was to to and transductants were to medium with and to this medium were of phage Botstein D. PubMed Scopus Google Scholar). DNA was isolated from strains using a Madison, DNA was into to that of the phenotype with of the for of into S. typhimurium LT2 were as reported G.A. Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). cobB + were identified by their to medium with and EcoRI fragment of plasmid pCOBB1 was cloned into the site of plasmid pSU19 E. Bartolome B. de la Cruz F. Gene (Amst.). 1988; 68: 159-162Crossref PubMed Scopus (253) Google Scholar) with This plasmid A of plasmid pCOBB2 the element was by P22 grown was to +) to of element the plasmid from medium containing and The new plasmid is to as plasmid SalI fragment of plasmid pCOBB2 was cloned into the site of plasmid pSU19 with This plasmid pCOBB4 was and The fragment was cloned into plasmid pSU19 with the The fragment of plasmid pCOBB5 was cloned into the overexpression vector with the +) was by into which plasmid rpo The was grown at in broth containing kanamycin and to a cell density of × were for in a with by at cultures were in an at cultures were were by × at for to of the tetA gene were to sequence the flanking of the was and was pCOBB4 DNA was isolated coli using Madison, F. S. S. A. PubMed Scopus Google Scholar) was to sequence using S. as were as the sequence flanking element was This was to the sequence of the region containing The nucleotide sequence of cobB is from the accession number cultures of grown overexpression were by × at for with a and extracts were by using a this were in and at at a of for extracts were to for the first was by at at using a In vitro described by Trzebiatowski and Escalante-Semerena (4Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1997; 272: 17662-17667Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) for the activity of the CobT protein were to for PRTase activity in cell-free extracts enriched for extracts containing high of the CobB enzyme (Fig. were from a a of cobT and the overexpression plasmid The reaction NaMN specific activity cell-free of in a of The reaction was at for and by to for protein was at at × for in a and in the were by using a system. The product of the reaction, was at the and was from Me2Bza and of α-ribazole-5′-phosphate was performed with a Molecular concentrations were determined by the of M.J. J. Gen. PubMed Scopus Google Scholar) and PubMed Scopus Google Scholar). were by using the PubMed Scopus Google Scholar) and by with J. Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. Wiley Interscience, New Scholar). In these of the the activity of the enzyme. This enzyme the of to H. 1973; Scopus Google Scholar). of in was restored the medium was with or of grown in nutrient broth was to CobB could α-ribazole-5′-phosphate from NaMN and Me2Bza in were in of and medium D.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) with and A of the reaction was the and the was at A of a was elsewhere the as a of a of Me2Bza was the as A total of were and point presented in Table the of cobB mutations of cobT mutants of S. + cobT was to from and it a The that strains with this phenotype were isolated for the existence of an alternative phosphoribosyltransferase enzyme that can compensate for the lack of CobT in the assembly of the nucleotide loop of cobalamin in cobT mutants (2Trzebiatowski J.R. O'Toole G.A. Escalante-Semerena J.C. J. Bacteriol. 1994; 176: 3568-3575Crossref PubMed Google Scholar, 4Trzebiatowski J.R. Escalante-Semerena J.C. J. Biol. Chem. 1997; 272: 17662-17667Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). mutation was into the TR6583 + the was to from in the of This that cobB function be required for biosynthesis however, a of a mutation in cobB the time of cultures of was in the medium the times of strains and Table of cobB time of the medium is described + cobB + The of the medium is described Open table in a new tab previously reported that insertions in cobB S. typhimurium from using propionate as Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar). the phenotype of isolated mutants to this phenotype was to of the insertions a gene of All point mutants and that the lack of and of a gene 3′ to was for of the This to be of plasmid pCOBB5 the cobB + into its was to the cell to from in the of Me2Bza and to propionate as the of and located to the 27-centisome region of the using and J. Bacteriol. 1992; 174: PubMed Google Scholar) crosses that cobB was with genetic in the 27-centisome was with was with was for the of the gene order of cobB and pepT using The of of or in can be as the product of the gene order pepT were consistent with which cobB 3′ to whose location to pepT and is known in coli M. J. J. B. 1997; PubMed Scopus Google Scholar) and S. typhimurium J. Bacteriol. 1993; 175: PubMed Google of cobB to of of selected (Cmr) transductants were selected medium containing and a of the at The order was cobB pepT + cobB transductants were selected medium containing and a of the at The order was cobB pepT The Open table in a new tab + cobB The nucleotide sequence of cobB and the predicted primary sequence of the protein is shown in in the location of have the sequence located from the as the site (also known as sequences are located and from the the and for cobB were identified and these sequences were by the sequence is to the sequence of the sequence is from the sequence be that the and sequences are and their in cobB to be that the analysis of the cobB sequence showed that this gene its in plasmid pCOBB1 which was isolated from the gene vector this plasmid was isolated as of the of cobB we that of the cobB was the of an within the was to such a Computer of the cobB and CobB sequences with gene and protein sequence to of known cobB CobB showed to cobT or The in E. however, is as a member of the SIR2 family of M. J. J. B. 1997; PubMed Scopus Google Scholar). The that the of these is are in the location of the in S. typhimurium and E. In E. coli the potABCD operon and the cobB are by in S. typhimurium the sequence these is 81 that the region to cobB be in S. To the biochemical analysis of the CobB the of CobB in the cell was to ∼30% of the total protein by cobB the of a phage T7 and site (Fig. extracts from the were to CobB NaMN:Me2Bza PRTase presented in A and a the of CobB in the and the synthesis of α-ribazole-5′-phosphate (Fig. In containing cell-free of strains that the overexpression plasmid to α-ribazole-5′-phosphate as by the lack of of the cobB (Fig. As reaction lacking a of protein to of the these that CobB has PRTase a of activity using CobB protein is The gene the alternative enzyme for CobT has been physically and the in it that cobB is and of an of the genetic and biochemical presented in this paper would be a new NaMN:Me2Bza phosphoribosyltransferase enzyme that is specific for and as with phosphoribosylpyrophosphate for NaMN in the W. and J. C. Escalante-Semerena, CobB has this it is as an enzyme as CobB can the cell requirement for cobalamin for the of CobB phosphoribosyltransferase activity is the for cobalamin is or as and A would that CobB is an enzyme, it is required for the synthesis of the alternative phosphoribosyltransferase enzyme. This is discussed have previously reported the requirement of CobB for the catabolism of propionate in S. typhimurium Escalante-Semerena J.C. J. Bacteriol. PubMed Google Scholar). specific biochemical activity for CobB in this has been identified we that a lack of cobB function transcription of the A. A. and Escalante-Semerena, J. C. J. Bacteriol. in which the enzymes for the of propionate in this Escalante-Semerena J.C. J. Bacteriol. 1997; PubMed Scopus Google Scholar). R. and J. C. Escalante-Semerena, for This is for the The CobB protein to be a member of the SIR2 family of eucaryotic transcriptional regulators. CobB the andYTQNID (Fig. which are a to the sequences and sequences are diagnostic of this family of regulatory 1995; PubMed Scopus Google Scholar). is that in to these the the sequence a region of high these are are is is and is The number of and in this region would be consistent with the that members of this family of to have a in this The identification of CobB as a member of the SIR2 family of would be consistent with the of CobB the of In light of this we are the that CobB have the it be required for the of the alternative PRTase enzyme. of the activity of CobB This is in The of PRTase activity in regulatory is The protein of is a member of a family of that transcription by to F. M.R. A. Full Text PDF PubMed Scopus Google Scholar, M. M. A. A. J. Bacteriol. PubMed Google Scholar, P. C. S. A. 1993; PubMed Scopus Google Scholar, J. P. S. A. 1993; PubMed Scopus Google Scholar, P. Mol. 1994; PubMed Scopus Google Scholar, M. M. S. J. 1997; PubMed Scopus Google Scholar). is a it is this activity is for the regulatory function of In the of the phosphoribosyltransferase activity of in B. is at this activity is J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this an alternative enzyme by the gene J. P. J. Bacteriol. 1995; 177: PubMed Google Scholar). has been that from an PRTase in which and were and an Smith Full Text Full Text PDF PubMed Scopus Google Scholar). presented shows that the PRTase activity of CobB is CobB be the first PRTase with regulatory J. Trzebiatowski for and C. and E. for