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

Trzebiatowski, Jodi R.; Escalante‐Semerena, Jorge C.

doi:10.1074/jbc.272.28.17662

Cited by 56 publications

(83 citation statements)

References 29 publications

(27 reference statements)

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“…The authors further show that the attachment of adenine to form pseudo-B 12 occurs by the same pathway as the attachment of DMB to form B 12 [DMB]. This result confirms previous biochemical findings that the nucleotide activation and attachment enzymes accommodate a variety of substrates but prefer DMB (4,29). The genetic evidence that pseudo-B 12 functions as a cofactor for all three B 12 -dependent enzymes in S. enterica also suggests that the formation of pseudo-B 12 is a natural physiological process (29).…”

supporting

confidence: 80%

“…One intriguing possibility is that none exist in S. enterica. This possibility was obscured in previous studies by the presence of DMB in agar, coupled with the 15-fold-higher activity of the nucleotide activation enzyme CobT with DMB compared to that with adenine (29). It is curious that S. enterica produces only ϳ100 molecules per cell of B 12 [DMB] when DMB is not provided exogenously (1,2).…”

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confidence: 75%

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Pseudo-B ₁₂ Joins the Cofactor Family

Taga

Walker

2008

J Bacteriol

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confidence: 80%

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confidence: 75%

Pseudo-B ₁₂ Joins the Cofactor Family

Taga

Walker

2008

J Bacteriol

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“…Protein samples were analyzed by SDS-PAGE (18) and stained with Coomassie blue (26). The N-terminal sequence of purified protein samples was performed at the Protein/Nucleic Acid Shared Facility at the Medical College of Wisconsin (Milwaukee, Wis.) as described previously (33).…”

Section: Vol 182 2000 Dihydroflavin-dependent Reduction Of Cobalamimentioning

confidence: 99%

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

Fonseca

Escalante‐Semerena

2000

J Bacteriol

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Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His- The biologically active, coenzymic form of cobalamin (Cbl) contains a 5Ј-deoxyadenosyl (Ado) group as the upper axial ligand (Fig. 1). Formation of the C-Co bond between the cobalt ion and the upper ligand requires that the cobalt ion be reduced to its ϩ1 oxidation state. Reduction of Co(III) to Co(I) is thought to proceed in two consecutive one-electron reductions catalyzed by cob(III)alamin reductase (EC 1.6.99.8) and by cob(II)alamin reductase (EC 1.6.99.9) (36) (Fig. 2). The product of cob(II)alamin reductase, a Co(I) corrinoid, is the substrate for the ATP:co(I)rrinoid adenosyltransferase (CobA) (EC 2.5.1.17) enzyme that generates the C-Co bond. This bond is very labile and needs to be reformed to maintain activity. Hence, this branch of the Ado-Cbl biosynthetic pathway is essential for coenzyme recycling.In Salmonella enterica serovar Typhimurium LT2, the CobA enzyme responsible for the last step of the pathway has been isolated and partially characterized (30, 31). Although enzymic activities that can generate reduced corrinoids have been reported and in some cases isolated, the genes encoding the enzymes responsible for the reductive steps of the pathway have not been identified in any organism (2,15,36).Reduction of cob(III)alamin by crude cell-free extracts of Clostridium tetanomorphum and Propionibacterium freundenreichii showed an absolute requirement for added flavin cofactors (3,36). In these bacteria and in Pseudomonas denitrificans, NADH was the preferred source of electrons for the reaction (2). A system that included thiol compounds, a diaphorase enzyme, NADH, and flavin adenine dinucleotide (FAD) was used to reduce Cbl in P. freundenreichii (3,15). Interestingly, this complex-reducing system was replaced by FADH 2 when purified preparations of the adenosyltransferase from this organism were used. The cob(II)yrinic acid a,c-diamide reductase enzyme of P. denitrificans was isolated and shown to require the addition of flavin cofactors for activity (2). The purified enzyme was shown to reduce complete and incomplete Co(II) corrinoids. Here again, the gene encoding this activity was not identified.Extensive efforts to isolate mutants of S. enterica serovar Typhimurium LT2 defective in corrinoid reduction and adenosylation have failed to identify genetic loci other than cobA (9; M. V. Fonseca and J. C. Escalante-Semerena, unpublished results). Hence, we took a reverse genetics approach to the isolation of ...

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“…DMB-independent growth required the CobT enzyme ( Fig. 4B and D), which is known to be necessary for the addition of DMB as an ␣-axial ligand (30,55).…”

Section: Conditions That Prevent Synthesis or Installation Of Any ␣-Amentioning

confidence: 99%

One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B ₁₂ Cofactors in Salmonella enterica

et al. 2008

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Coenzyme B 12 (Ado-Cbl) and vitamin B 12 (CN-B 12 ) (Fig. 1) include the base 5,6-dimethylbenzimidazole (DMB) as an ␣-axial ligand. Prokaryotes, the only producers of vitamin B 12 , make "complete" corrinoid cofactors with a variety of alternative ␣-axial ligands, including benzimidazoles, phenols, and purines (16, 52). These alternative corrinoids may be functional equivalents of coenzyme B 12 for many bacterial enzymes, but functional differences are suggested by the selectivity of the human assimilatory protein, intrinsic factor for cobalamin, the DMB-containing vitamin B 12 (53).Under strictly anaerobic growth conditions, Salmonella enterica synthesizes corrinoids de novo (44) and installs either adenine (to form pseudo-coenzyme B 12 [Ado-pseudo-B 12 ]) or 2-methyl-adenine (to form adenosyl-factor A) as an ␣-axial ligand (22). If even trace amounts of oxygen are present, DMB-containing B 12 (cobalamin [Cbl]) is also made (39). When grown aerobically on glucose, S. enterica cannot synthesize the corrin moiety, which must be supplied as an "incomplete" corrinoid, such as cobinamide (Cbi), with its corrin ring and aminopropanol side chain. Under these conditions, coenzyme B 12 is made, with DMB as the ␣-axial ligand (20). Figure 2 diagrams the de novo (anaerobic) synthetic pathway and assimilation of exogenous Cbi or . Notice that the ␤-ligand adenosyl can be added (by CobA) to three different substrates and that the (CN) 2 Cbi supplied as the corrin ring source is not a normal CobA substrate.Here, the origins and installation of ␣-axial ligands are approached genetically using an unexpected feature of S. enterica. While S. enterica can use exogenous Cbi to produce B 12 cofactors (cobalamins) during aerobic growth on glucose (20), it makes only about 100 molecules per cell (2). This is apparently insufficient B 12 coenzyme to support growth on ethanolamine (5) unless the DMB base is also supplied. That is, under these conditions, cells neither make sufficient DMB nor install an alternative ligand to allow corrinoid-dependent aerobic growth on ethanolamine (5).This situation allowed positive selection of mutants that can grow on ethanolamine plus Cbi without added DMB. These mutants were expected to show either increased endogenous DMB production or to install an alternative base as an ␣-axial ligand. All of the mutants made pseudo-B 12 cofactors, which have adenine as an ␣-axial ligand, and most of these mutations affected purine metabolism so as to increase the intracellular level of free adenine base. The same set of enzymes (CobUSTC) installs either adenine base (to form pseudo-B 12 ) or DMB (to form vitamin B 12 ). A model suggests how the choice is made. MATERIALS AND METHODSBacterial strains and transposons. Strains were derived from S. enterica (serovar Typhimurium) LT2

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

Cited by 56 publications

References 29 publications

Pseudo-B ₁₂ Joins the Cofactor Family

Pseudo-B ₁₂ Joins the Cofactor Family

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B ₁₂ Cofactors in Salmonella enterica

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

Cited by 56 publications

References 29 publications

Pseudo-B 12 Joins the Cofactor Family

Pseudo-B 12 Joins the Cofactor Family

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B 12 Cofactors in Salmonella enterica

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Product

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Pseudo-B ₁₂ Joins the Cofactor Family

Pseudo-B ₁₂ Joins the Cofactor Family

One Pathway Can Incorporate either Adenine or Dimethylbenzimidazole as an α-Axial Ligand of B ₁₂ Cofactors in Salmonella enterica