Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.
Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.
Debussche
L
L
Département Analyse, Institut des Biotechnologies, Vitry-sur-Seine, France.
Couder
M
M
Thibaut
D
D
Cameron
B
B
Crouzet
J
J
Blanche
F
F
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D014578:Uroporphyrins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000096:biosynthesis*
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D014578:Uroporphyrins; D003035:Cobalt; C078111:hydrogenobyrinic acid-a,c-diamide; D008190:Lyases; C033211:cobaltochelatase; D014805:Vitamin B 12