Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans.
The cobalamin biosynthetic pathway enzyme that catalyzes amidation of 5'-deoxy-5'-adenosyl-cobyrinic acid a,c-diamide was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by a four-column procedure. The purified protein had an isoelectric point of 5.6 and molecular weights of 97,300 as estimated by gel filtration and 57,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. Stepwise Edman degradation provided the sequence of the first 16 amino acid residues at the N terminus. The enzyme catalyzed the four-step amidation sequence from cobyrinic acid a,c-diamide to cobyric acid via the formation of cobyrinic acid triamide, tetraamide, and pentaamide intermediates. The amidations are carried out in a specific order; this order was not determined. The enzyme was specific to coenzyme forms of substrates and did not carry out amidation of the carboxyl group at position f. The amidation reactions were ATP/Mg2+ dependent and exhibited a broad optimum around pH 7.5. L-Glutamine was shown to be the preferred amide group donor (Km congruent to 45 microM) but could be replaced by ammonia (Km = 20 mM). For all of the four partially amidated substrates, the Km values were in the micromolar range and the Vmax values were about 7,000 nmol h-1 mg-1.
Département Analyse, Institut des Biotechnologies, Vitry-sur-Seine, France.
F
F
Blanche
M
M
Couder
L
L
Debussche
D
D
Thibaut
B
B
Cameron
J
J
Crouzet
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D003038:Cobamides / Q000096:biosynthesis / Q000378:metabolism*; D005784:Gene Amplification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014805:Vitamin B 12 / Q000096:biosynthesis* / Q000378:metabolism
D000577:Amides; D003038:Cobamides; D014805:Vitamin B 12