Searching for Intermediates in the Carbon Skeleton Rearrangement of 2-Methyleneglutarate to (R)-3-Methylitaconate Catalyzed by Coenzyme B12-Dependent 2-Methyleneglutarate Mutase from
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- 作者：Antonio J. Pierik ; Daniele Ciceri ; Ruben Fernandez Lopez ; Fritz Kroll ; Gerd Brö ; ker ; Birgitta Beatrix ; Wolfgang Buckel ; and Bernard T. Golding
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 9, 2005
- 年：2005
- 卷：44
- 期：31
- 页码：10541 - 10551
- 全文大小：170K
- 年卷期：v.44,no.31(August 9, 2005)
- ISSN：1520-4995
文摘
Coenzyme B12-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacteriumbarkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins withmutations in the highly conserved coenzyme binding-motif DXH(X)2G(X)41GG (D483N and H485Q)exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings areconsistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand ofadenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all fourstereoisomers 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, Ki = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR)signal (gxy 
2.1; gz 2.0), which by analogy with the findings on glutamate mutase from Clostridiumcochlearium [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 concomitantwith the formation of oxygen-insensitive cob(II)alamin (gxy 2.25; gz 2.0). In order to identify thecarbon-centered radical, various 13C- and one 2H-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 of2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-13C]adenosylcobalaminwas used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-eliminationand fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase.
2.1; gz 2.0), which by analogy with the findings on glutamate mutase from Clostridiumcochlearium [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 concomitantwith the formation of oxygen-insensitive cob(II)alamin (gxy 2.25; gz 2.0). In order to identify thecarbon-centered radical, various 13C- and one 2H-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 of2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-13C]adenosylcobalaminwas used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-eliminationand fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase.