Publications

• On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri.

  Hartrampf G., Buckel W.

  The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which w ... >> More

  The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face. 2-Methylene[2-2H1]glutarate was synthesized via (R)-3-methyl[3-2H1]itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O. The predominantly monodeuterated compound was oxidized to (S)-[2-2H1]succinate as analysed by circular dichroism. The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon. << Less

  Eur J Biochem 156:301-304(1986) [PubMed] [EuropePMC]

• Cloning, sequencing and expression of the gene encoding the coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri in Escherichia coli.

  Beatrix B., Zelder O., Linder D., Buckel W.

  The coenzyme B12 (adenosylcobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri. (a) The mgm gene encoding 2-methylene ... >> More

  The coenzyme B12 (adenosylcobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri. (a) The mgm gene encoding 2-methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8-kDa protein of 614 amino acids. It shows significant similarity in its C-terminal part to that of other cobamide-dependent enzymes. Probably, this is the coenzyme-binding region. (b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western-blot analysis with rabbit antiserum directed against the native mutase. (c) Cell-free extracts from E. coli carrying the mgm gene showed 2-methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme. << Less

  Eur. J. Biochem. 221:101-109(1994) [PubMed] [EuropePMC]

• On the role of two different cobalt(II) species in coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri.

  Zelder O., Buckel W.

  Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enz ... >> More

  Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enzyme liberated the prosthetic groups, cob(II)alamin being oxidized by air to aquocobalamin. HPLC analysis of the released cobamides from several preparations revealed > 90% adenosylcobalamin and < 10% aquocobalamin. Treatment of active 2-methyleneglutarate mutase with 8M urea followed by gelfiltration yielded an inactive enzyme from which 50% of the adenosylcobalamin and up to 70% of the cob(II)alamin was removed. Addition of adenosylcobalamin to the urea-treated enzyme resulted in complete reactivation, but the content of cob(II)alamin was not increased. These data suggest that the oxygen-stable cob(II)alamin is not involved in catalysis. In the presence of the competitive inhibitor itaconate (methylenesuccinate, Ki = 0.7mM), an alteration of the UV/visible spectrum at 470 nm as well as a new line in the EPR spectrum of the enzyme (around g = 2.1) was observed. The results indicate the formation of an unusual, oxygen sensitive Co(II) species during catalysis. The EPR signal of the oxygen-stable cob(II)alamin (gx,y = 2.24) remained unchanged under those conditions. << Less

  Biol. Chem. Hoppe-Seyler 374:85-90(1993) [PubMed] [EuropePMC]

• Searching for intermediates in the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalyzed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri.

  Pierik A.J., Ciceri D., Lopez R.F., Kroll F., Broker G., Beatrix B., Buckel W., Golding B.T.

  Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited d ... >> More

  Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings are consistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand of adenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all four stereoisomers of 1-methylcyclopropane-1,2-dicarboxylate were synthesized and tested, along with acrylate, with respect to their inhibitory potential. Acrylate and the 2-methylpent-2-enedioates were noninhibitory. Among the 1-methylcyclopropane-1,2-dicarboxylates only the (1R,2R)-isomer displayed weak inhibition (noncompetitive, K(i) = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR) signal (g(xy) approximately 2.1; g(z) approximately 2.0), which by analogy with the findings on glutamate mutase from Clostridium cochlearium [Biochemistry, 1998, 37, 4105-4113] was assigned to cob(II)alamin coupled to a carbon-centered radical. At longer incubation times (>1 h), inactivation of the mutase occurred concomitant with the formation of oxygen-insensitive cob(II)alamin (g(xy) approximately 2.25; g(z) approximately 2.0). In order to identify the carbon-centered radical, various (13)C- and one (2)H-labeled substrate/product molecules were synthesized. Broadening (0.5 mT) of the EPR signal around g = 2.1 was observed only when C2 and/or C4 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-(13)C]adenosylcobalamin was used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-elimination and fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase. << Less

  Biochemistry 44:10541-10551(2005) [PubMed] [EuropePMC]

• Assay and purification of the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri.

  Michel C., Hartrampf G., Buckel W.

  A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much hi ... >> More

  A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (delta epsilon = 3.7 mM-1.cm-1). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (delta epsilon = 4.0 mM-1.cm-1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amine-Sepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I = 0.1 M and 25 degrees C: K1, app = [(R)-3-methylitaconate].[2-methyleneglutarate]-1 = 0.26 +/-0.04, K2,app = [2,3-dimethylmaleate].[(R)-3-methylitaconate]-1 = 7.40 +/- 0.21. << Less

  Eur. J. Biochem. 184:103-107(1989) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.