Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography

Blanche, Francis; Thibaut, D; Couder, M; Muller, Jean-Claude

doi:10.1016/0003-2697(90)90038-b

Cited by 42 publications

(34 citation statements)

References 20 publications

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“…With no standard of Ado-cobyrinic acid for HPLC, this latter result was demonstrated as follows. During HPLC analysis of incubations with cobyrinic acid, all radioactive peaks were collected, treated with cyanide, and shown to be free of cobyrinic acid (4). Unlike other corrinoid precursors of cobalamin, which were quantitatively reduced under the incubation conditions described above, cobyrinic acid was not quantitatively reduced to the Co(I) state, as determined spectrophotometrically.…”

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confidence: 98%

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans

et al. 1991

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confidence: 98%

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans

et al. 1991

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“…The following compounds were obtained as described previously: [1,11,17-methyl-14C]precorrin-6x (compound 2b in Fig. 1) (24), hydrogenobyrinic acid and hydrogenobyrinic acid a,c-diamide (13), (CN)2cobyrinic acid (6), and (aq)2cobyrinic acid (13).…”

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confidence: 99%

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate

et al. 1992

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The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. The recent isolation of precorrin-6x (24), a biosynthetic precursor of hydrogenobyrinic acid (compound 5a in Fig. 1) (8,20), and the elucidation of the structure of precorrin-6x octamethyl ester (compound 2c in Fig. 1) (22) have dramatically changed the direction of research on the biosynthesis of vitamin B12. This discovery has modified our views concerning the order of the biochemical events required for the synthesis of the corrin macrocycle and has permitted a clear proposal for the biosynthetic processes occurring during the conversion of precorrin-6x (compound 2a in Fig. 1) into hydrogenobyrinic acid. In particular, this study suggested the involvement of an NADPH-dependent reduction of precorrin-6x as the first enzymatic reaction (Fig. 2) (24). Conclusive evidence for this reduction step was recently gained when precorrin-6y (compound 3 in Fig.

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“…Hydrophobic chromatography. Active fractions from step 4 (eluted with 0.12 M potassium chloride) were pooled and concentrated to 14 ml in Centriprep 10 concentrators (Amicon). Samples of this solution (3.5 ml each) were mixed with 1.…”

Section: Methodsmentioning

confidence: 99%

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene

et al. 1992

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Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050Bacteriol. 174: -1052Bacteriol. 174: , 1992 in the presence of [methyl-3HIS-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr -27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 ± 0.2 ,uM for precorrin-6x and 23.5 ± 3.5 ,uM for NADPH and a V.x value of 17,000 U mg-' were obtained at pH 7.7.The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment

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Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography

Cited by 42 publications

References 20 publications

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene

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