Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester.

Thibaut, D; Blanche, Francis; Debussche, Laurent; Leeper, Finian J.; Battersby, Alan R.

doi:10.1073/pnas.87.22.8800

Cited by 54 publications

(27 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4A). This protein had a molecular weight of 33,000, which was in good agreement with the molecular weight predicted for the fused protein (33,509). Similarly, pXL1874 contained a gene fusion between the first three codons of lacZ and a truncated cobT missing the first three codons; the expression of this fusion was under the control of Plac.…”

Section: Cgcggcgcgaacagcggcgcatcctgatgatgatttcggacggcgcgccggtcgacgactsupporting

confidence: 81%

Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes

Cameron¹,

Guilhot²,

Blanche³

et al. 1991

J Bacteriol

Self Cite

View full text Add to dashboard Cite

A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertionmediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.Cobalamin biosynthesis is considered to require between 20 and 30 steps to transform uroporphyrinogen III into cobalamin. Most of the steps for the conversion of uroporphyrinogen III into cobyrinic acid remain to be established, but they include at least eight C methylations, elimination of C-20, NADPH-dependent reduction of the macrocycle, decarboxylation of the C-12 acetate, methyl migration from C-li to C-12, and cobalt insertion (for details, see references 23 and 33). Based on the study of Pseudomonas denitrificans, an up-to-date cobalamin biosynthesis scheme is presented in the accompanying paper (12).We have published data on the cloning of at least 14 cob genes (7), which are grouped into four clusters on the P. denitrificans chromosome. Genetic and biochemical analyses allowed the identification of 13 cob genes: cobA to cobE on a 5.4-kb fragment from complementation group C (11) and cobF to cobM on an 8.7-kb fragment from complementation group A (10). With the objective of searching for and identifying all the cob genes, a detailed genetic study of the complementation groups has been undertaken. In particular, our investigation focused on a large DNA fragment which was reported to contain two complementation classes (7). We present here the genetic analysis of a 12-kb P. denitrificans DNA fragment from complementation group B (7) and the nucleotide sequence of a 4.8-kb subfragment. Only two cob genes were found. The steps in the cobalamin pathway at which the encoded Cob proteins might act were investigated.

show abstract

Section: Cgcggcgcgaacagcggcgcatcctgatgatgatttcggacggcgcgccggtcgacgactsupporting

confidence: 81%

Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes

Cameron¹,

Guilhot²,

Blanche³

et al. 1991

J Bacteriol

Self Cite

View full text Add to dashboard Cite

show abstract

“…pXL1908, which contains the ORF3 3' end, ORF4, cobO, and ORF6, complemented these mutants (Fig. 4) (4,5,23,34,40,44,46). Between precorrin-3 and cobyrinic acid, the order of the steps is not known except for the methylations; however, the structure of precorrin-6x (44,46) indicates that reduction of the macrocycle, methylations at C-5 and C-15, decarboxylation at C-12, and methyl migration from C-il to C-12 occur last.…”

Section: Resultsmentioning

confidence: 83%

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

Crouzet¹,

Lévy-Schil²,

Cameron³

et al. 1991

J Bacteriol

Self Cite

122

View full text Add to dashboard Cite

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,

show abstract

“…The methyl ester derivative exhibited Xmax (CH2Cl2) at 336 nm (log e 4.36, sh), 349 nm (4.40), and 381 nm (3.91, sh), with Xmin at 287 nm (3.40). The spectrum of this pigment was therefore very similar to that of precorrin-6x (19), except that it was =20-nm shortwave shifted. This indicated that the molecule had, like precorrin-6x, separated chromophores with a methyl group still at C-11.…”

Section: Resultsmentioning

confidence: 66%

“…More recently, a previously undescribed biosynthetic precursor of hydrogenobyrinic acid, named precorrin-6x, was isolated from P. denitrificans (22), and the complete structure of its methylester derivative was established (compound 2c in Fig. 1) (19).…”

mentioning

confidence: 99%

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene

et al. 1992

Self Cite

View full text Add to dashboard Cite

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050Bacteriol. 174: -1052Bacteriol. 174: , 1992 in the presence of [methyl-3HIS-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr -27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 ± 0.2 ,uM for precorrin-6x and 23.5 ± 3.5 ,uM for NADPH and a V.x value of 17,000 U mg-' were obtained at pH 7.7.The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment

show abstract

Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester.

Cited by 54 publications

References 15 publications

Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes

Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene

Contact Info

Product

Resources

About