THE ROLE OF TWO π-STACKED TRYPTOPHAN RESIDUES IN OXALATE DECARBOXYLASE

Anthony Pastore, Umar Twahir, Matt Burg, Steve Bruner, and Alexander Angerhofer

Department of Chemistry, University of Florida, Gainesville FL 32611-7200, USA

Oxalate Decarboxylase (OxDC) is a bicupin enzyme from Bacillus subtilis that is natively expressed in the cytoplasm and also for the coats of endospores. It crystallizes as a hexamer. Each cupin (beta barrel) domain coordinates a Mn ion for a total of two Mn per monomer - with one Mn binding site near the N-terminal and another near the C-terminal. OxDC is of interest because it can process oxalate through two pathways; a significant decarboxylation pathway to produce carbon dioxide and formate, as well as a minor oxidase pathway to create hydrogen peroxide and carbon dioxide. Both pathways depend on the presence of dioxygen. The oxidase pathway consumes dioxygen while it appears to be a co-catalyst in the decarboxylase pathway by generating Mn(III) which may be the main driving force for catalysis. The binding site of dioxygen on the enzyme is still unclear. If it binds in the N-terminal Mn-binding pocket, the active site, it is unclear how the decarboxylase pathway is mechanistically possible. If it binds at the C-terminal Mn binding site then a long-range electron transfer between the two Mn ions is necessary for activity. This might be facilitated by a pair of π-stacked tryptophan residues, W96 and W274. Some aspects of the mechanism for OxDC remain elusive, however, one hypothesis stated that long range electron transfer - or hopping - is necessary for catalysis. To test this hypothesis OxDC mutants W96F, W274F, W96Y, W274Y, and the corresponding double mutants have been expressed. The poster presents the crystal structure of W96F together with other data from the mutants that lend tentative support for the importance of both tryptophans for catalysis.