Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase

Abstract

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 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).…”

Membrane topology of Escherichia coli diacylglycerol kinase

“…Protein and oligonucleotide sequence searches made by using the BLAST algorithm (1) uncovered four new known or probable prokaryotic DAGK sequences, which are listed in Fig. 2 (6,18,22,29,36). Examination of the manually aligned sequences shows that four of the five exhibit fairly divergent sequences (e.g., <40% identity between E. coli DAGK and the DAGK from the other organisms).…”

ReddishEscherichia coliCells Caused by Overproduction ofBacillus stearothermophilusUroporphyrinogen III Methylase: Cloning, Sequencing, and Expression of the Gene

“…7), which encodes nicotinate-nucleotide dimethyl benzimidazole phosphoribosyltransferase, participating in the cobalamin biosynthesis. 28) The sequence homology strongly suggested that the 28-kDa protein is an uroporphyrinogen III methylase. The enzymic activity was assayed and the results are presented in Table III.…”