Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Nov 1992; 174(22): 7445–7451.
PMCID: PMC207441

Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.

Abstract

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 denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides 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 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)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 (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Berhauer K, Wagner F, Michna H, Rapp P, Vogelmann H. Zur Chemie und Biochemie der Corrinoide, XXIX. Biogenesewege von der Cobyrinsäure zur Cobysäure und zum Cobinamid bei Propionibacterium shermanii. Hoppe Seylers Z Physiol Chem. 1968 Oct;349(10):1297–1309. [PubMed]
  • Blanche F, Debussche L, Famechon A, Thibaut D, Cameron B, Crouzet J. A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities. J Bacteriol. 1991 Oct;173(19):6052–6057. [PMC free article] [PubMed]
  • Blanche F, Debussche L, Thibaut D, Crouzet J, Cameron B. Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans. J Bacteriol. 1989 Aug;171(8):4222–4231. [PMC free article] [PubMed]
  • Blanche F, Maton L, Debussche L, Thibaut D. Purification and characterization of Cob(II)yrinic acid a,c-diamide reductase from Pseudomonas denitrificans. J Bacteriol. 1992 Nov;174(22):7452–7454. [PMC free article] [PubMed]
  • Blanche F, Thibaut D, Couder M, Muller JC. Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography. Anal Biochem. 1990 Aug 15;189(1):24–29. [PubMed]
  • Blanche F, Thibaut D, Famechon A, Debussche L, Cameron B, Crouzet J. Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene. J Bacteriol. 1992 Feb;174(3):1036–1042. [PMC free article] [PubMed]
  • Cameron B, Blanche F, Rouyez MC, Bisch D, Famechon A, Couder M, Cauchois L, Thibaut D, Debussche L, Crouzet J. Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase. J Bacteriol. 1991 Oct;173(19):6066–6073. [PMC free article] [PubMed]
  • Cameron B, Briggs K, Pridmore S, Brefort G, Crouzet J. Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans. J Bacteriol. 1989 Jan;171(1):547–557. [PMC free article] [PubMed]
  • Cameron B, Guilhot C, Blanche F, Cauchois L, Rouyez MC, Rigault S, Levy-Schil S, Crouzet J. Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes. J Bacteriol. 1991 Oct;173(19):6058–6065. [PMC free article] [PubMed]
  • Crouzet J, Cameron B, Cauchois L, Rigault S, Rouyez MC, Blanche F, Thibaut D, Debussche L. Genetic and sequence analysis of an 8.7-kilobase Pseudomonas denitrificans fragment carrying eight genes involved in transformation of precorrin-2 to cobyrinic acid. J Bacteriol. 1990 Oct;172(10):5980–5990. [PMC free article] [PubMed]
  • Crouzet J, Levy-Schil S, Cameron B, Cauchois L, Rigault S, Rouyez MC, Blanche F, Debussche L, Thibaut D. 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. J Bacteriol. 1991 Oct;173(19):6074–6087. [PMC free article] [PubMed]
  • Debussche L, Couder M, Thibaut D, Cameron B, Crouzet J, Blanche F. Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans. J Bacteriol. 1991 Oct;173(19):6300–6302. [PMC free article] [PubMed]
  • Debussche L, Thibaut D, Cameron B, Crouzet J, Blanche F. Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans. J Bacteriol. 1990 Nov;172(11):6239–6244. [PMC free article] [PubMed]
  • Djalali M, Gueant JL, Lambert D, el Kholty S, Saunier M, Nicolas JP. High-performance liquid chromatographic separation and dual competitive binding assay of corrinoids in biological material. J Chromatogr. 1990 Jul 13;529(1):81–91. [PubMed]
  • Dresow B, Schlingmann G, Ernst L, Koppenhagen VB. Extracellular metal-free corrinoids from Rhodopseudomonas spheroides. J Biol Chem. 1980 Aug 25;255(16):7637–7644. [PubMed]
  • Fenton WA, Rosenberg LE. The defect in the cbl B class of human methylmalonic acidemia: deficiency of cob(I)alamin adenosyltransferase activity in extracts of cultured fibroblasts. Biochem Biophys Res Commun. 1981 Jan 15;98(1):283–289. [PubMed]
  • Labbe-Bois R. The ferrochelatase from Saccharomyces cerevisiae. Sequence, disruption, and expression of its structural gene HEM15. J Biol Chem. 1990 May 5;265(13):7278–7283. [PubMed]
  • Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed]
  • Schrauzer GN, Deutsch E, Windgassen RJ. The nucleophilicity of vitamin B12. J Am Chem Soc. 1968 Apr 24;90(9):2441–2442. [PubMed]
  • Thibaut D, Blanche F, Debussche L, Leeper FJ, Battersby AR. Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8800–8804. [PMC free article] [PubMed]
  • Thibaut D, Couder M, Crouzet J, Debussche L, Cameron B, Blanche F. Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans. J Bacteriol. 1990 Nov;172(11):6245–6251. [PMC free article] [PubMed]
  • Thibaut D, Couder M, Famechon A, Debussche L, Cameron B, Crouzet J, Blanche F. The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate. J Bacteriol. 1992 Feb;174(3):1043–1049. [PMC free article] [PubMed]
  • Thibaut D, Debussche L, Blanche F. Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining five S-adenosylmethionine-derived peripheral methyl groups. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8795–8799. [PMC free article] [PubMed]
  • Toohey JI. A vitamin B12 compound containing no cobalt. Proc Natl Acad Sci U S A. 1965 Sep;54(3):934–942. [PMC free article] [PubMed]
  • Toohey JI. Cobalt-free corrinoid compounds from photosynthetic bacteria. Fed Proc. 1966 Nov-Dec;25(6):1628–1632. [PubMed]
  • Walker CJ, Weinstein JD. Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts : substrate specificity, regulation, intactness, and ATP requirements. Plant Physiol. 1991 Apr;95(4):1189–1196. [PMC free article] [PubMed]
  • Walker CJ, Weinstein JD. In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5789–5793. [PMC free article] [PubMed]
  • Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. [PMC free article] [PubMed]
  • WEISSBACH H, LADD JN, VOLCANI BE, SMYTH RD, BARKER HA. Structure of the adenylcobamide coenzyme: degradation by cyanide, acid, and light. J Biol Chem. 1960 May;235:1462–1473. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...