Structural and biochemical studies of Streptococcus sp. alpha-glycerophosphate oxidase.

摘要

The ability for bacteria to grow on glycerol has been linked to the genes responsible for encoding the glycerol facilitator (GlpF), glycerol kinase (GlpK), and either alpha-glycerophosphate oxidase (GlpO) or dehydrogenase (GlpD). GlpF aids in bringing glycerol into the cell, while GlpK phosphorylates it, trapping it within the cell as alpha-glycerophosphate (Glp). Glp is converted to dihydroxyacetone phosphate (DHAP) by GlpO in certain bacteria and by GlpD in others. Heme deficient lactic acid bacteria, such as Streptococcus sp. and Enterococcus casseliflavus, contain GlpO, whereas bacteria such as Bacillus subtilis and Escherichia coli contain GlpD. Both GlpO and GlpD utilize flavin adenine dinucleotide (FAD) as a cofactor, which is concomitantly reduced upon substrate oxidation. Though sequentially similar, GlpOs and GlpDs are functionally different based on the reoxidation of the cofactor; GlpO reacts with O2 to form H2O2, while GlpD transfers the reducing equivalents to ubiquinone. GlpO and GlpD also differ in that GlpDs are considered membrane associated, while most GlpOs are considered cytosolic. The GlpOs from heme deficient lactic acid bacteria also contain a ca. 50-residue insert that is not present in GlpDs. Limited proteolysis experiments with the Streptococcus sp. GlpO enzyme have suggested that this insert region may be a flexible surface loop region.;A 2.4 A resolution x-ray crystal structure of the streptococcal GlpO enzyme has been determined, along with a 2.3 A resolution x-ray crystal structure of a deletion mutant (GlpODelta) lacking the 50-residue insert region but retaining catalytic activity. A comparison of these two structures shows conformational changes involving an active-site histidine (H65) and the isoalloxazine ring of FAD. This has been interpreted as being representative of two forms of the resting oxidized enzyme, seen biochemically with the enterococcal GlpO enzyme.;Comparisons of GlpODelta with two structural homologs, D-amino acid oxidase (DAAO) and glycine oxidase (ThiO), showed the presence of a structurally conserved arginine residue, R346 in GlpODelta. Further analysis of the active site of G1pODelta resulted in the finding of other residues that are conserved among the GlpOs and GlpDs and are also within proposed Glp-binding regions, namely H65, R69, Y70, and K429; however, these residues are not present in DAAO or ThiO. This has led to the hypothesis that these residues form a phosphate binding pocket. An active-site base is required for the abstraction of a proton from the C(2)-OH of Glp and drives the hydride transfer of C(2)-H to FAD N(5). Histidines are common residues that play this role in oxidoreductases. A hypothesis was developed that H65 may play that role.;Mutagenic analyses show that R346 and K429 are essential for GlpO activity. Both residues when mutated yielded an inactive enzyme based on the horseradish peroxidase (HRP) assay and enzyme-monitored turnover (EMT) experiments. Sulfite titration data for both mutants revealed that they both stabilized an FAD N(5)-sulfite adduct, though at a higher Kd when compared to wild-type GlpODelta. Analyzing the relationship of Kd values for sulfite and redox potential, the increase in sulfite Kd seems to rule out the mutations affecting the redox potentials of the FAD cofactor for either of these mutants. Based on the HRP assay data, H65A is ∼2% as active as wild type GlpODelta, whereas the H65Q mutant is ∼63% as active. This result contradicts the hypothesis that H65 is an active site base. This result, however, does support the idea that this residue is important for activity but for substrate binding, possibly interacting with the Glp-phosphate moiety. The activity for the R69M is ∼13% that of wild type, implicating it as an important residue in catalysis, also possibly interacting with the Glp-phosphate upon substrate binding. The Y70F mutant yields an activity of ∼77% of wild type, indicating that it may be important for enzyme catalysis but not essential. The structural and biochemical experiments described in this dissertation have led to the formulation of a reaction scheme for binding and catalysis of GlpO.