A 13.1-kb DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contained five different cob genes named cobN to cobQ and cobW. Based on the similarity of NH2-terminal sequences and molecular weights of the purified Cob proteins, CobQ was identified as cobyric acid synthase, CobP was identified as a bifunctional enzyme exhibiting both cobinamide kinase and cobinamide phosphate guanylyltransferase activities, and CobO was identified as cob(I)alamin adenosyltransferase. CobN is proposed to play a role in cobalt insertion reactions. Four other open reading frames were identified on the 13.1-kb fragment, but their chromosomal inactivation did not lead to a cobalamin-minus phenotype.The cobalamin biosynthetic pathway probably involves 20 to 30 different enzymatic steps (Fig. 1) consisting of (i) formation of uroporphyrinogen III (urogen III), which is the common intermediate for the synthesis of hemes, chlorophylls, cobalamins, F430, and sirohemes; (ii) conversion of urogen III into cobyrinic acid by three successive methylations at C-2, C-7, and C-20 followed by five other methylations at C-17, C-11, C-1, C-5, and C-15, decarboxylation of the acetic acid side chain at C-12, elimination of C-20, NADPH-dependent reduction of the macrocycle, methyl migration from C-11 to C-12, and insertion of cobalt; (iii) formation of cobinamide from cobyrinic acid (Fig. 1) by amidation of the peripheral carboxyl groups a, b, c, d, e, and g and insertion of (R)-l-amino-2-propanol at position f; and (iv) conversion of cobinamide into coenzyme B12 (for reviews on cobalamin synthesis, see references 4, 5, 23, 34, and 40). In addition, the central cobalt atom is adenosylated to form the coenzyme derivatives. We have described elsewhere the cloning of Pseudomonas denitrificans genes involved in cobalamin synthesis (cob genes) (12) from urogen III. These genes are grouped into four genomic loci that correspond to four distinct complementation groups (from A to D). We have reported the nucleotide sequence and the results of genetic analysis of a 5.4-kb DNA fragment from complementation group C carrying five cob genes (cobA to cobE) elsewhere (16). This fragment is located on the right end of the complementation group C restriction map (Fig. 2). cobA and cobB are the structural genes for S-adenosyl-L-* Corresponding author. t Present address: 5 bis rue Pierre Curie,

A 5.4-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced, The nucleotide sequence and genetic analysis revealed that this fragment carries five different cob genes (cobA to cobE). Four of these genes present the characteristics of translationally coupled genes. cobA has been identified as the structural gene of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) because the encoded protein has the same NH2 terminus and molecular weight as those determined for the purified SUMT. For the same reasons the cobB gene was shown to be the structural gene for cobyrinic acid-a,c-diamide synthase. Genetic and biochemical data concerning cobC and cobD mutants suggest that the products of these genes are involved in the conversion of cobyric acid to cobinamide.The cobalamin biosynthetic pathway probably involves 20 to 30 different enzymatic steps, consisting of (i) formation of uroporphyrinogen III (urogen III), which is the common intermediate for the synthesis of hemes, chlorophylls, cobalamins, F430, and sirohemes; (ii) conversion of urogen III into cobyrinic acid, including the methylations at C-1, C-2, C-5, C-7, C-12, C-15, C-17, and C-20, the decarboxylation of the acetic side chain at C-12, the loss of C-20, and the introduction of cobalt; (iii) formation of cobinamide from cobyrinic acid by amidation of six of seven peripheral carboxylic groups, the seventh being amidated by (R)-1-amino-2-propanol; (iv) conversion of cobinamide into coenzyme B12 (for reviews on cobalamin synthesis, see references 3, 4, 18, 28, and 39). Only one enzymatic activity involved in the transformation of urogen III to coenzyme B12 has been purified (7), and no biosynthetic intermediate has been purified along the precorrin-3-to-cobyrinic-acid pathway. Cloned genes involved in cobalamin synthesis (cob genes) are valuable tools for the study of the coenzyme B12 biosynthesis at the biochemical and genetic levels. These genes should enable the identification of enzymatic activities and biosynthetic intermediates of the pathway and facilitate the understanding of the nature of biochemical and genetic regulation mechanisms operative in the cob regulon.We have reported the cloning of at least 14 different genes, involved in cobalamin biosynthesis in Pseudomonas denitrificans, based on complementation data (9). Of these 14 genes, 12 are involved in the transformation of urogen III into cobinamide. The other two complement Cob mutants blocked in the conversion of cobinamide into cobalamin and * Corresponding author. are implicated in the last four steps of the cobalamin biosynthetic pathway (18). All 14 cloned genes are grouped on the P. denitrificans genome in four genomic regions, corre. sponding to complementation groups A, B, C, and D (9). In contrast, most of the cob genes in Salmonella typhimuriumand Bacillus megaterium are clustered (23,24, 45). We report the genetic analysis and nucleotide sequence of a 5.4-kilobase-pair (kb) fragment from complementation group C. Part of this fragment is...

A 8.7-kilobase DNA fragment carrying Pseudomonas denitrificans cob genes has been sequenced. The nucleotide sequence and the genetic analysis revealed that this fragment carries eight different cob genes (cobF to cobM). Six of these genes have the characteristics of translationafly coupled genes. cobl has been identified as S-adenosyl-L-methionine (SAM):precorrin-2 methyltransferase structural gene because the encoded protein has the same NH2 terminus and mholecular weight as those of the purified enzyme. From protein homology with CobA and CobI, two SAM-dependent methyltransferases of the cobalamin pathway, it is proposed that cobF, cobJ, cobL, and cobM code for other methyltransferases involved in the cobalamin pathway. In addition, purified CobF protein has affinity for SAM, as expected for a SAM-dependent methyltransferase. Accumulation of cobalamin precursors in Agrobacterium tumefaciens mutants complemented by any of these eight genes suggest that, apart from cobI, whose function is identified, the products of all these genes are implicated in the conversion of precorrin-3 into cobyrinic acid.

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).

A BacUlus megatenium DNA fragment encoding S-adenosyl-L-methionine:uroporphyrinogen Im methyltransferase (SUMT) activity was subcloned and sequenced. The encoded polypeptide showed more than 43.5% strict homology to Pseudomonas denitrificans SUMT (F. Blanche, L. Debussche, D. Thibaut, J. Crouzet, and B. Cameron, J. Bacteriol. 171:4222-4231, 1989). The B. megaterium polypeptide was overexpressed in Escherichia coli, partially purified, and shown to exhibit, like P. denirficans SUMT, substrate inhibition at uroporphyrinogen m concentrations above 0.5 ,uM, suggesting a common regulation for aerobic cobalaminproducing organisms.S-Adenosyl-L-methionine (SAM):uroporphyrinogen III (urogen III) methyltransferase (SUMT), a key enzyme in the coenzyme B12 pathway, is the first enzyme to occur after the branching of the cobalamin and heme pathways. It catalyzes the transfer of two methyl groups from SAM to urogen III at C-2 and C-7 to give precorrin-2, a common intermediate in corrinoids, siroheme, and F430 synthesis (9, 14). Two enzymes having SUMT activity have already been studied. The Pseudomonas denitrificans enzyme is encoded by the cobA gene, and P. denitrificans mutants deficient in SUMT activity have a cobalamin-minus phenotype (1). The P. denitrificans enzyme is a homodimer of Mr 2x 29,200 with a low turnover number and an activity that exhibits a substrate inhibition phenomenon at urogen III concentrations above 2 ,uM (1). The Escherichia coli cysG product is involved in the synthesis of siroheme and has a molecular weight of 49,900 (17). This protein has been found to catalyze the same activity as the P. denitrificans CobA protein (20). Both proteins show more than 40%o strict homology on most of the CobA primary sequence (5). However, the CysG protein has an NH2-terminal domain (more than 200 amino acids) whose function has been tentatively proposed (5) to be associated with the transformation of precorrin-2 into siroheme (involving oxidation and iron chelation). We are interested in understanding the regulation of the cobalamin pathway and in improving the productivity of strains used for cobalamin production. Enzymes with SUMT activity are of great interest, since their activities, in competition with that of urogen III decarboxylase, determine the metabolic flux entering the cobalamin pathway rather than the heme pathway. We have chosen to study the SUMT structural gene from Bacillus megaterium, a strict aerobe that produces high levels of cobalamins (8) and in which the genetics of cobalamin synthesis have been studied (4).We used plasmid pGX3512 (the generous gift of R. N. Brey [3]), which corresponds to pBR322 (2) with a 4-kb Sau3AI B. megaterium fragment inserted into the BamHI site. This plasmid complemented the E. coli cysG44 muta-* Corresponding author. t Present address: