Enzymic Synthesis of Guanosine Diphosphate Cobinamide by Extracts of Propionic Acid Bacteria<sup>*</sup>

Ronzio, Robert A.; Barker, H. A.

doi:10.1021/bi00860a009

Cited by 31 publications

(19 citation statements)

References 23 publications

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“…3, Panel A, lane 2). This prominent band was absent from extracts of a control strain (JE3420), which carried only the overexpression vector pT7-7 ( The relative mobility of radiolabeled (CN) 2 CBI-P on the chromatograph was 0.77, a value consistent with that reported in the literature (15). This radioactive product comigrated with authentic (CN) 2 CBI-P; unlabeled (CN) 2 CBI-P was identified on the chromatogram by its characteristic red-purple color (data not shown).…”

Section: Overexpression and Visualization Of Cobusupporting

confidence: 86%

Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

O’Toole

Escalante‐Semerena

1995

Journal of Biological Chemistry

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The CobU protein of Salmonella typhimurium was overexpressed and purified to ϳ94% homogeneity. Nterminal sequencing of purified CobU confirmed the first 22 amino acids. In vitro assays showed that CobU has kinase and guanylyltransferase activities which catalyze the synthesis of adenosyl-cobinamide-GDP from adenosyl-cobinamide, via an adenosyl-cobinamidephosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosylcobinamide-phosphate proceeds via an phosphoramidate-linked, enzyme-guanylyl intermediate.In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost. Treatment of inactive CobU with dithiothreitol restored ϳ20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents 5,5-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide abolished both CobU activities.Native CobU protein was a dimer (ϳ40 kDa) that functioned optimally at pH 8.8 -9.0 and 37°C. Substrates and kinetic parameters for both activities were determined. The preferred corrinoid substrate for this enzyme was adenosyl-cobinamide. In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen.

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Section: Overexpression and Visualization Of Cobusupporting

confidence: 86%

Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

O’Toole

Escalante‐Semerena

1995

Journal of Biological Chemistry

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“…(CN)2CBI-P was used as the starting material for the synthesis of (CN)2CBI-GDP by the procedure of Kennedy (20) as reported by Ronzio and Barker (25) without modifications. (CN)2CBI-GDP was further purified by RP-HPLC on a pBondapak C18 (10-pm packing; 3.9 by 300 mm) steel column (Waters) developed by a modification of the system reported elsewhere (5) as follows: the flow rate was maintained during the run at 1 ml min-1, and the column was equilibrated with a buffer system containing 1% solvent A, 98% solvent B, and 1% solvent C (see below).…”

Section: Resultsmentioning

confidence: 99%

Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin

1993

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The CoblIl region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis of the nucleotide loop of CBL and comprises three genes, designated cobU, cobS, and cobT (26). Complementation studies identified two classes of CobI1 mutants: (i) 34 mutants were complemented by a plasmid carrying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmid carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were isolated. These data were consistent with those of complementation studies done with F' cobUST plasmids, which also suggested that the CobIli region comprises two complementation groups.A plasmid carrying cobUS' was sufficient to complement a deletion of the entire CobIII region, a result suggesting that CobT was not required for CBL biosynthesis. Nutritional studies done with synthetic putative intermediates of the CobIll pathway were performed to further classify cobIII mutants. A subset of cobU mutants were found to be responsive to exogenous dicyano-cobinamide-GDP, while cobS mutants were found to be responsive only to CBL. These results are consistent with the adenosyl-cobinamide kinase-GTP:adenosylcobinamide-phosphate guanylyltransferase and CBL synthase activities proposed for CobU and CobS, respectively. The cobIII genes under the control of the T7 promoter were overexpressed, and the resulting polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides with apparent molecular masses of 22, 26, and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected.

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“…The present work provides the first evidence that both cobinamide kinase and cobinamide phosphate guanylyltransferase activities are specific to the coenzyme forms of their respective substrates. Studies on P. shermanii also indicated that both activities require a thiol-reducing agent (16,18). The enzyme from P. denitrificans did not show such a requirement during purification or storage, or during the assay of activities.…”

Section: Discussionmentioning

confidence: 97%

“…The Very few studies on the enzymes involved in these latest steps of the cobalamin pathway have been reported to date. In vitro enzymatic synthesis of cobinamide phosphate by cobinamide kinase (reaction 1) (16,17) and of GDP-cobinamide by cobinamide phosphate guanylyltransferase (reaction 2) (17,18) has been demonstrated with crude protein extracts of cobalamin-producing microorganisms, such as P. * Corresponding author.…”

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confidence: 99%

A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities

Blanche¹,

Debussche²,

Famechon³

et al. 1991

J Bacteriol

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Enzymic Synthesis of Guanosine Diphosphate Cobinamide by Extracts of Propionic Acid Bacteria^*

Cited by 31 publications

References 23 publications

Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin

A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities

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Enzymic Synthesis of Guanosine Diphosphate Cobinamide by Extracts of Propionic Acid Bacteria*

Cited by 31 publications

References 23 publications

Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

Purification and Characterization of the Bifunctional CobU Enzyme of Salmonella typhimurium LT2

Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin

A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities

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Enzymic Synthesis of Guanosine Diphosphate Cobinamide by Extracts of Propionic Acid Bacteria^*