Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase

Abstract

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,

The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium

“…2 (27). The sequence of these genes is homologous with that of genes of Pseudomonas denitrificans that have been shown to encode enzymes catalyzing reactions involved in joining adenosyl-CBI and DMB (2,4,5,9). These sequences and their analysis will be presented in detail elsewhere (11).…”

“…Genetic studies indicate that the CobII region is composed of four complementation groups. Correlation of the genetic map with available DNA sequence data for the CobI and CobIII regions of the operon suggests that three open reading frames are included in the CobIII region (5,(7)(8)(9)11).…”

Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans

“…These studies suggested that the insertion of the adenosyl group takes place at the stage of cobyrinic acid a,c-diamide. In addition, cobO mutants G632 and G633 (7,8) have been shown to accumulate cobyrinic acid a,c-diamide specifically (28 and 4% of the amount of cobalamin produced by the parent strain, respectively), and the present study has revealed the enzymatic activity and substrate specificity of the cobO gene product. We infer from these data that the main physiological function of CobO is to adenosylate cob(I) yrinic a,c-diamide.…”

“…An elevated adenosyltransferase activity (about 3 to 6 U mg of protein-' versus 0.1-0.3 U mg-1) was detected in SC510 Rif' strains containing plasmid pXL227, which carries the cobO gene (8). SC510 Rif(pXL227) was used as the source of the enzyme for purification.…”

A Putative Mg Chelatase Subunit from Arabidopsis thaliana cv C24 (Sequence and Transcript Analysis of the Gene, Import of the Protein into Chloroplasts, and in Situ Localization of the Transcript and Protein

“…CHL H also exhibits 65% identity with ChlH, a putative Mg chelatase subunit from Synechocystis PCC 6803 (Jensen et al, 1996a). Hudson et al (1993) noted the strong similarity between the OLI protein and CobN, which is a subunit of the cobaltochelatase enzyme from Pseudomonas dentrificans (Crouzet et al, 1991). Similarly, the Arabidopsis CHL H protein shows 27% identity with CobN.…”