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

Debussche, Laurent; Couder, M; Thibaut, D; Cameron, Béatrice; Crouzet, J; Blanche, Francis

doi:10.1128/jb.173.19.6300-6302.1991

Cited by 47 publications

(30 citation statements)

References 16 publications

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“…(iii) Insertion of the Ado group presumably takes place at the stage of cobyrinic acid a,c-diamide. Our study of P. denitrificans cob(I)alamin adenosyltransferase fully confirmed this latter conclusion (10). The obligatory role of cobyric acid in cobalamin biosynthesis (Fig.…”

Section: Discussionsupporting

confidence: 77%

Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans

Blanche¹,

Couder²,

Debussche³

et al. 1991

J Bacteriol

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The cobalamin biosynthetic pathway enzyme that catalyzes amidation of 5'-deoxy-5'-adenosyl-cobyrinic acid a,c-diamide was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by a four-column procedure. The purified protein had an isoelectric point of 5.6 and molecular weights of 97,300 as estimated by gel filtration and 57,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. Stepwise Edman degradation provided the sequence of the first 16 amino acid residues at the N terminus. The enzyme catalyzed the four-step amidation sequence from cobyrinic acid a,c-diamide to cobyric acid via the formation of cobyrinic acid triamide, tetraamide, and pentaamide intermediates. The amidations are carried out in a specific order; this order was not determined. The enzyme was specific to coenzyme forms of substrates and did not carry out amidation of the carboxyl group at positionf. The amidation reactions were ATP/Mg2+ dependent and exhibited a broad optimum around pH 7.5. L-Glutamine was shown to be the preferred amide group donor (Km = 45 ,uM) but could be replaced by ammonia (Km = 20 mM). For all of the four partially amidated substrates, the Km values were in the micromolar range and the Vmax values were about 7,000 nmol h-1 mg-'.

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Section: Discussionsupporting

confidence: 77%

Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans

Blanche¹,

Couder²,

Debussche³

et al. 1991

J Bacteriol

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“…Hence, co(II)rrin reduction could be coupled with the adenosylation of the corrin. In support of this idea, we have shown that CobR appears to interact with the B. melitensis CobO (the cob(I)yrinic acid a,c-diamide adenosyltransferase (36,37), which is sometimes referred to as CobA or BtuR (38,39)) through the increase in fluorescence polarization observed on the titration of CobO into a solution of CobR (supplemental Fig. S6).…”

Section: Discussionmentioning

confidence: 63%

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

Lawrence

Deery

McLean

et al. 2008

Journal of Biological Chemistry

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Vitamin B 12 , the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6 Å resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction. Cobalamin (vitamin B 12 ) is a coenzyme that is associated with a range of isomerization, methylation, and dehalogenation reactions, utilizing the unique chemistry of the carbon-cobalt bond that is characteristic of this molecule (1). The octahedral geometry of the functional cobalt ion in cobalamin necessitates a structurally complex molecule that consists of a corrin ring, a lower nucleotide loop, and an upper ligand that is either a methyl group in methylcobalamin or an adenosyl group in adenosylcobalamin. The corrin ring is a modified tetrapyrrole and as such bears a structural resemblance to molecules such as heme, chlorophyll, siroheme, and coenzyme F 430 , reflecting a common synthesis of the tetrapyrrole template as part of a broader branched metabolic pathway (2). However, cobalamin differs from these other modified tetrapyrroles in two important aspects. First, the carbon framework of the corrin molecule has been contracted such that one of the integral carbons of the tetrapyrrole progenitor has been removed to afford a smaller central cavity into which the cobalt ion is bound. Second, cobalamin provides both upper and lower ligands for the cobalt to complement the ligands provided by the nitrogens from the four pyrrole-derived units of the macrocycle.Central to the role played in catalysis by cobalamin-containing enzymes is the cobalt ion, which forms a unique cobaltcarbon bond with either an upper adenosyl (adenosylcobalamin) or methyl (methylcobalamin) group. It is the physical properties of this bond that mediate the scintillating chemistry associated with B 12

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“…The position of the cobalt insertion-mediating reactions is discussed in the text (** indicates that cobalt insertion takes place on hydrogenobyrinic acid a,c-diamide. The adenosylation reaction is shown to occur on cobyrinic acid a,c-diamide (18); therefore, all the following intermediates are adenosylated. Identified enzymes: 1, S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase; 2, S-adenosyl-L-methionine:precorrin-2 methyltransferase; 3, cobyrinic a,c-diamide synthase; 4, cob(I)alamin adenosyltransferase; 5, cobyric acid synthase; 6, cobinamide kinasecobinamide phosphate guanylyltransferase; 7, cobalamin (5'-phosphate) synthase, which uses either 5,6-dimethylbenzimidazole-ribose (DBIR) or 5,6-dimethylbenzimidazole-ribose-5'-phosphate (DBIRP) as a substrate (11); 8, nicotinate-nucleotide:dimethylbenzimidazole phosphoribosyltransferase.…”

Section: Resultsmentioning

confidence: 99%

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

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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,

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

Cited by 47 publications

References 16 publications

Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans

Biosynthesis of vitamin B12: stepwise amidation of carboxyl groups b, d, e, and g of cobyrinic acid a,c-diamide is catalyzed by one enzyme in Pseudomonas denitrificans

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

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