The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.
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. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.
Thibaut
D
D
Département Analyse, Centre de Recherche de Vitry-Alfortville, Vitry-sur-Seine, France.
Couder
M
M
Famechon
A
A
Debussche
L
L
Cameron
B
B
Crouzet
J
J
Blanche
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002474:Cell-Free System; D019751:Intramolecular Transferases; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D014578:Uroporphyrins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D014578:Uroporphyrins; C072475:precorrin 8x; C072476:hydrogenobyrinic acid; D009249:NADP; D007535:Isomerases; D019751:Intramolecular Transferases; C072474:CobH protein, Pseudomonas denitrificans