Abstract
The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9 A resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit-subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products.