Structural studies of enzymes involved in drug resistance and antibiotic biosynthesis.

摘要

The emergence of pathogenic bacteria resistant to the current battery of effective antibiotics remains a global health concern. Antibiotic resistance genes are plasmid borne and can easily be transferred between pathogens, regardless of phylogeny. The macrolide antibiotic erythromycin can be enzymatically degraded by strains carrying the ereB locus that codes for erythromycin esterase type B and confers high level resistance to this widely used antibiotic. We have determined high resolution crystal structures of apo-EreB, EreB bound to its final product and an inactive EreB variant bound to the substrate erythromycin. In addition, we also performed biochemical studies on EreB such as HPLC tracing of erythromycin degradation, agar plate based bioassay and enzyme-substrate affinity based on isothermal titration calorimetry. Our structural and biochemical data helps to characterize EreB and elucidate its catalytic mechanism, providing a valuable framework to develop efficient inhibitors of EreB for pharmaceutical application.;Lipoglycopeptide antibiotics, such as teicoplanin and A40926, have shown efficacy against bacteria that are resistant to the traditional battery of antibiotics. Pseudoaglycone deacetylase plays a crucial role in the maturation process of these antibiotics. These enzymes remove an acetyl group from the pseudoaglycone prior to the acylation reaction that yields the final product. The pseudoaglycone deacetylases in the biosynthesis of teicoplanin and A40926 were identified as Orf2* and Dbv21, respectively. We have determined the crystal structures of each of these enzymes, Orf2* bound to one of its products myristic acid and the inhibited form of Orf2* in the presence of excess of zinc ions. These studies should provide a framework in the enzyme engineering for the development of novel, potent antimicrobial reagents.;Lipopolysaccharide (LPS) is important for the persistence and pathogenesis of Gram-negative bacteria. Nudix enzyme GDP-mannose mannosyl (Gmm) hydrolase helps to maintain the sugar diversity of lipopolysaccharide by negatively regulating the concentration of GDP-α-D-mannose in the biosynthetic pathway of O-antigen, such that plays an important role in the biological function of LPS. We determined the structures of apo-Gmm and Gmm bound to different products and substrates. These high resolution crystal structures provide framework to reconcile previous kinetic parameters and help to address the molecular basis of substrate selectivity and specificity. In addition, these structures reveal a concerted conformational change along the active site upon ligand binding.