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 denitrijia. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of Mr 140,000 and 450,000, which were purified to homogeneity. The 140,000-Mr component was shown to be coded by cobN, whereas the 450,000-Mr component was composed of two poWpeptides 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 ,uM, respectively. Spectroscopic data revealed that the reaction product was cob(ll)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

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an M r of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with M r s of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with M r s estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an M r of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.

TnS Spr transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide:dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P.denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and a-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and a-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.Conversion of cobinamide into cobalamin requires four enzymatic steps in Propionibacterium shermanii (17,24). First, phosphorylation of the hydroxyl group of the (R)-1-amino-2-propanol residue of cobinamide leads to cobinamide phosphate (reaction A). Second, addition of the GMP moiety of a molecule of GTP onto cobinamide phosphate generates GDP-cobinamide (reaction B). Third, exchange of GMP with a-ribazole 5'-phosphate within GDP-cobinamide yields cobalamin 5'-phosphate (reaction C). Fourth, dephosphorylation of cobalamin 5'-phosphate gives cobalamin (reaction D).

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.

No abstract