Mechanism, structure and inhibition of UDP-galactopyranose mutase.

摘要

Galactofuranose residues are crucial cell wall components in many microbial pathogens, including Mycobacterium tuberculosis . The biosynthetic precursor of galactofuranose residues is UDP-galactofuranose, which is produced by the flavoenzyme UDP-galactopyranose mutase (UGM). As galactofuranose residues are not found in mammals, UGM represents an attractive target for antimicrobial agents. To better understand this enzyme, I investigated its mechanism and structure. I also tested the consequences of its inhibition.;Flavoenzymes are widely known for redox catalysis, but UGM is one of the relatively few that catalyzes a non-redox reaction. We proposed that turnover proceeds through a covalent flavin-galactose iminium ion intermediate. In support of this mechanism, I trapped this putative intermediate with sodium cyanoborohydride. The adduct can be isolated in high yield, and its structure is consistent with the predicted N5-alkylflavin species.;UGM catalysis was studied further by using X-ray crystallography to determine the structure of the enzyme complexed with the substrate analog UDP-glucose. Consistent with the proposed mechanism, the ligand binds adjacent to the active site. The sugar portion of the ligand, however, is not aligned for covalent catalysis. Indeed, this orientation may be important for UGM discrimination against UDP-glucose.;To determine how UGM binds its substrate, I soaked crystals of the UGM--UDP-glucose complex in UDP-galactopyranose. The structure of the enzyme-substrate complex, in both oxidized and reduced forms, was solved by X-ray crystallography. The uridine portions of the substrate and non-substrate (UDP-glucose) bind similarly, while the positions of the sugar moieties differ. Although substrate binds in a non-productive manner in the oxidized structure, chemical reduction of the substrate-bound crystal results in a reorientation of the substrate such that it is poised to engage in covalent catalysis.;Inhibition of UGM was also investigated. In prokaryotic UGMs, a secondary binding site near the active site was identified, explaining the tight binding of inhibitory fluorescent probes to UGM. I also identified the first inhibitors of a eukaryotic UGM (from Aspergillus fumigatus). These compounds, selected for activity from those that block prokaryotic UGMs, have potencies (400-1100 nM IC50 values) that exceed those of any previously described UGM inhibitors.