Structure of α-glycerophosphate oxidase from Streptococcus sp.:: A template for the mitochondrial α-glycerophosphate dehydrogenase

The FAD-dependent alpha-glycerophosphate oxidase (GlpO) from Enterococcus casseliflavus and Streptococcus sp. was originally studied as a soluble flavoprotein oxidase; surprisingly, the G1pO sequence is 30-43% identical to those of the alpha-glycerophosphate dehydrogenases (G1pDs) from mitochondrial and bacterial sources. The structure of a deletion mutant of Streptococcus sp. G1pO (G1pO Delta, lacking a 50-residue insert that includes a flexible surface region) has been determined using multiwavelength anomalous dispersion data and refined at 2.3 angstrom resolution. Using the G1pO Delta structure as a search model, we have also determined the intact G1pO structure, as refined at 2.4 angstrom resolution. The first two domains of the GIpO fold are most closely related to those of the flavoprotein glycine oxidase, where they function in FAD binding and substrate binding, respectively; the GIpO C-terminal domain consists of two helix bundles and is not closely related to any known structure. The flexible surface region in intact G1pO corresponds to a segment of missing electron density that links the substrate-binding domain to a beta beta alpha element of the FAD-binding domain. In accordance with earlier biochemical studies (stabilizations of the covalent FAD-N5 - sulfite adduct and p-quinonoid form of 8-mercapto-FAD), Ile430-N, Thr431-N, and Thr431-OG are hydrogen bonded to FAD-O2 alpha in G1pO Delta, stabilizing the negative charge in these two modified flavins and facilitating transfer of a hydride to FAD-N5 (from G1p) as well. Active-site overlays with the glycine oxidase-N-acetylglycine and D-amino acid oxidase-D-alanine complexes demonstrate that Arg346 of G1pO Delta is structurally equivalent to Arg302 and Arg285, respectively; in both cases, these residues interact directly with the amino acid substrate or inhibitor carboxylate. The structural and functional divergence between G1pO and the bacterial and mitochondrial G1pDs is also discussed.