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Lookup NU author(s): Antonius Pierik, Daniele Ciceri, Emeritus Professor Bernard Golding
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Coenzyme B12-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barken catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations 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 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, Ki = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-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 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 (gxy ≈ 2.25; gz ≈ 2.0). In order to identify the carbon-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 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5′-13C] 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. © 2005 American Chemical Society.
Author(s): Pierik AJ, Ciceri D, Lopez RF, Kroll F, Broker G, Beatrix B, Buckel W, Golding BT
Publication type: Article
Publication status: Published
Journal: Biochemistry
Year: 2005
Volume: 44
Issue: 31
Pages: 10541-10551
ISSN (print): 0006-2960
ISSN (electronic): 1520-4995
Publisher: American Chemical Society
URL: http://dx.doi.org/10.1021/bi050049n
DOI: 10.1021/bi050049n
PubMed id: 16060663
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