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.
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.
Pierik
Antonio J
AJ
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany.
Ciceri
Daniele
D
Lopez
Ruben Fernandez
RF
Kroll
Fritz
F
Bröker
Gerd
G
Beatrix
Birgitta
B
Buckel
Wolfgang
W
Golding
Bernard T
BT
D002244:Carbon / Q000737:chemistry*; D002384:Catalysis; D003038:Cobamides / Q000138:chemical synthesis*; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry; D005051:Eubacterium / Q000201:enzymology* / Q000235:genetics; D005977:Glutarates / Q000138:chemical synthesis*; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013386:Succinates / Q000138:chemical synthesis*
D003038:Cobamides; D004791:Enzyme Inhibitors; D005977:Glutarates; D013386:Succinates; C048646:3-methylitaconate; C002172:2-methyleneglutaric acid; D002244:Carbon; D019751:Intramolecular Transferases; C047997:2-methyleneglutarate mutase; C000913:cobamamide