A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane. (1/71)

Methylobacterium sp. strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.  (+info)

Methanol:coenzyme M methyltransferase from Methanosarcina barkeri -- substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin. (2/71)

Methyl-coenzyme M formation from coenzyme M and methanol in Methanosarcina barkeri is catalysed by an enzyme system composed of three polypeptides MtaA, MtaB and MtaC, the latter of which harbours a corrinoid prosthetic group. We report here that MtaC can be substituted by free cob(I)alamin which is methylated with methanol in an MtaB-catalysed reaction and demethylated with coenzyme M in an MtaA-catalysed reaction. Methyl transfer from methanol to coenzyme M was found to proceed at a relatively high specific activity at micromolar concentrations of cob(I)alamin. This finding was surprising because the methylation of cob(I)alamin catalysed by MtaB alone and the demethylation of methylcob(III)alamin catalysed by MtaA alone exhibit apparent Km for cob(I)alamin and methylcob(III)alamin of above 1 mm. A possible explanation is that MtaA positively affects the MtaB catalytic efficiency and vice versa by decreasing the apparent Km for their corrinoid substrates. Activation of MtaA by MtaB was methanol-dependent. In the assay for methanol:coenzyme M methyltransferase activity cob(I)alamin could be substituted by cob(I)inamide which is devoid of the nucleotide loop. Substitution was, however, only possible when the assays were supplemented with imidazole: approximately 1 mm imidazole being required for half-maximal activity. Methylation of cob(I)inamide with methanol was found to be dependent on imidazole but not on the demethylation of methylcob(III)inamide with coenzyme M. The demethylation reaction was even inhibited by imidazole. The structure and catalytic mechanism of the MtaABC complex are compared with the cobalamin-dependent methionine synthase.  (+info)

Native corrinoids from Clostridium cochlearium are adeninylcobamides: spectroscopic analysis and identification of pseudovitamin B(12) and factor A. (3/71)

The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co(beta)-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 micromol) of B(12) derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B(12) (Co(beta)-cyano-7"-adeninylcobamide) (60%) and factor A (Co(beta)-cyano-7"-[2-methyl]adeninylcobamide) (20%). Authentic pseudovitamin B(12) was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B(12) and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional (1)H, (13)C-, and (15)N nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-[N(6)-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native "complete" B(12) cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.  (+info)

The enigma of cobalamin (Vitamin B12) biosynthesis in Porphyromonas gingivalis. Identification and characterization of a functional corrin pathway. (4/71)

The ability of Porphyromonas gingivalis to biosynthesize tetrapyrroles de novo has been investigated. Extracts of the bacterium do not possess activity for 5- aminolevulinic-acid dehydratase or porphobilinogen deaminase, two key enzymes involved in the synthesis of uroporphyrinogen III. Similarly, it was not possible to detect any genetic evidence for these early enzymes with the use of degenerate polymerase chain reaction. However, the bacterium does appear to harbor some of the enzymes for cobalamin biosynthesis since cobyric acid, a pathway intermediate, was converted into cobinamide. Furthermore, degenerate polymerase chain reaction with primers to cbiP, which encodes cobyric-acid synthase, produced a fragment with a high degree of identity to Salmonella typhimurium cbiP. Indeed, the recently released genome sequence data confirmed the presence of cbiP together with 14 other genes of the cobalamin pathway. A number of these genes were cloned and functionally characterized. Although P. gingivalis harbors all the genes necessary to convert precorrin-2 into cobalamin, it is missing the genes for the synthesis of precorrin-2. Either the organism has a novel pathway for the synthesis of precorrin-2, or more likely, it has lost this early part of the pathway. The remainder of the pathway may be being maintained to act as a salvage route for corrin synthesis.  (+info)

The Na(+)-translocating methyltransferase complex from methanogenic archaea. (5/71)

Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA-H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions.  (+info)

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides. (6/71)

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.  (+info)

ABC transporter for corrinoids in Halobacterium sp. strain NRC-1. (7/71)

We report evidence for the existence of a putative ABC transporter for corrinoid utilization in the extremely halophilic archaeon Halobacterium sp. strain NRC-1. Results from genetic and nutritional analyses of Halobacterium showed that mutants with lesions in open reading frames (ORFs) Vng1370G, Vng1371Gm, and Vng1369G required a 10(5)-fold higher concentration of cobalamin for growth than the wild-type or parent strain. The data support the conclusion that these ORFs encode orthologs of the bacterial cobalamin ABC transporter permease (btuC; Vng1370G), ATPase (btuD; Vng1371Gm), and substrate-binding protein (btuF; Vng1369G) components. Mutations in the Vng1370G, Vng1371Gm, and Vng1369G genes were epistatic, consistent with the hypothesis that their products work together to accomplish the same function. Extracts of btuF mutant strains grown in the presence of cobalamin did not contain any cobalamin molecules detectable by a sensitive bioassay, whereas btuCD mutant strain extracts did. The data are consistent with the hypothesis that the BtuF protein is exported to the extracellular side of the cell membrane, where it can bind cobalamin in the absence of BtuC and BtuD. Our data also provide evidence for the regulation of corrinoid transport and biosynthesis. Halobacterium synthesized cobalamin in a chemically defined medium lacking corrinoid precursors. To the best of our knowledge, this is the first genetic analysis of an archaeal corrinoid transport system.  (+info)

Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri. (8/71)

Cobalamin and the native and diepimeric forms of factor F430 catalyzed the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene or chloroethane (CA) in a buffer with Ti(III) citrate as the electron donor. Ethylene was the major product in the cobalamin-catalyzed transformation, and the ratio of ethylene to CA formed was 25:1. Native F430 and 12,13-di-epi-F430 produced ethylene and CA in ratios of about 2:1 and 1:1, respectively. Cobalamin dechlorinated 1,2-DCA much faster than did factor F430. Dechlorination rates by all three catalysts showed a distinct pH dependence, correlated in a linear manner with the catalyst concentration and doubled with a temperature increase of 10 degrees C. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethylene and CA with Ti(III) citrate as the reductant. The catalytic components in boiled extracts were heat and oxygen stable and had low molecular masses. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic and part had a hydrophobic character. These chemical properties of the dechlorinating components and spectral analysis of boiled extracts indicated that corrinoids or factor F430 was responsible for the dechlorinations. The ratios of 3:1 to 7:1 of ethylene and CA formed by cell extracts suggested that both cofactors were concomitantly active.  (+info)

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