title: EPR spin trapping of an oxalate-derived free radical in the oxalate decarboxylase reaction.
abstract: EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly (13)C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution.
authors.affiliation: Department of Chemistry, The University of Florida, Gainesville, FL 32611–7200, USA.
authors.forename: Witcha
authors.initials: W
authors.lastname: Imaram
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Saylor
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Centonze
authors.forename: Nigel G J
authors.initials: NG
authors.lastname: Richards
authors.forename: Alexander
authors.initials: A
authors.lastname: Angerhofer
mesh_terms: D002262:Carboxy-Lyases / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D002262:Carboxy-Lyases; C021915:oxalate decarboxylase