Active site and loop 4 movements within human glycolate oxidase: implications for substrate specificity and drug design.

The human liver enzyme glycolate oxidase (GO1), also known as the HAOX1 gene product, is a member of the well-characterized FMN-dependent α-hydroxy acid oxidase enzyme family (1, 2). This family includes Pseudomonas putida mandelate dehydrogenase (MDH, 32% sequence identity), the flavin-binding domain of yeast flavocytochrome b2 (FCB2, 38%), rat long chain hydroxy acid oxidase (LCHAO, 74%), and spinach glycolate oxidase (GOX, 57%). Each enzyme exhibits the canonical β8/α8 fold and several conserved active site residues, suggestive of a common mechanism for the oxidation of the substrate and the reduction of the flavin ring in the first half reaction. The second half-reaction involves the transfer of electrons from the reduced flavin to either molecular oxygen or other electron acceptors including cytochromes. In contrast to GOX and the other enzymes, GO exhibits broad substrate specificity and is capable of oxidizing glycolate, glyoxylate, and long chain α-hydroxy acids (Figure 1) including 2-hydroxy octanoate (2-OH-8) and 2-hydroxy palmitate (2-OH-16) (3). Moreover, the substrate specificity of GO is markedly different from the kidney isozyme, HAOX2, a possible homologue of LCHAO. The ability of GO to oxidize glyoxylate to oxalate, a key metabolite in kidney stone formation, is of particular importance for individuals with primary hyperoxalaria type I, as a consequence of their inability to convert glyoxylate to glycine in the peroxisome (4). Figure 1 Substrates, products and an inhibitor of human GO. The progression of glycolate to oxalate via GO oxidation is indicated by arrows. Despite the overall similarity of the α-hydroxy acid oxidases, the variable sequences (Supporting Information (SI) Figure S1 online) and structures near the active site have been used to explain differences in activity and substrate preferences. In particular, the size of the amino acid side chain represented by position 110 of human GO has been hypothesized to determine the type of substrate oxidized (5–10). Another region of low sequence homology is found as an insert between β-strand β4 and α-helix α4 known as loop 4. Loop 4 has been shown to influence the activity of LCHAO, to provide a subdomain interface in FCB2, and to form a membrane-spanning domain of MDH (10–14). However, the variability in the visibility and conformation of loop 4 in the crystal structures of GOX, the chimeric MDH-GOX, lactate oxidase (LOX) from Aerococcus viridans, and LCHAO have made discerning the role of loop 4 in GO difficult (10, 14–17). How Trp110 and loop 4 may generate the substrate selectively of GO is not known since the crystal structure of the enzyme alone or in complex with ligands has not been described in the literature. In an effort to understand the structure–function relationships of human GO, we have determined the structure of recombinant GO in complex with sulfate, glyoxylate, and a potent inhibitor, 4-carboxy-5-dodecylsulfanyl-1,2,3-triazole (CDST), by X-ray crystallography. The kinetic parameters for the oxidation of glycolate, glyoxylate, and 2-OH-8 have also been determined to facilitate comparisons to other enzymes involved in determining the level of these metabolites and oxalate. The data presented herein provides insight into the role of GO in oxalate formation and the unique structural features of GO that may be exploited to develop therapeutic interventions for PH patients.