Poster abstracts
Poster number 59 submitted by Timothy Grunkemeyer
Deciphering the mechanism of the six-bladed β-propeller hydrolases
Timothy J. Grunkemeyer (OSBP), Dr. Kiran Doddapaneni (Department of Chemistry and Biochemistry, The Ohio State University), Srividya Murali (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Each year approximately 3 million people are exposed to toxic organophosphorous compounds (OPs), 10% of which succumb this exposure. This has led to the search for potential therapeutics in the form of bioscavengers. Several such bioscavengers are the closely related enzymes squid di-isopropylfluorophosphatase (DFPase), bacterial drug responsive protein 35 (Drp35), recombinant Paraoxonase-1 (PON1), and human senescence marker protein 30 (SMP30). These enzymes share the six-bladed β-propeller fold as well as promiscuous substrate specificities. However, due to a plethora of conflicting mechanistic evidence, their enzymatic mechanisms are poorly understood, thus limiting our protein engineering capabilities. In order to clarify the mechanisms, a comparative enzymology approach is being employed with DFPase, Drp35, PON1, and SMP30. We hypothesize that all four enzymes operate via a common mechanism. The wild type (WT) and critical active site mutants have been cloned and the expression and purification protocols have been optimized in a bacterial system. To interrogate their substrate tolerance, each construct has been screened against a panel of lactones, aryl esters, and OPs. All WT enzymes show strong lactonase activity while WT DFPase, PON1, and SMP30 will hydrolyze OPs, albeit with less specificity. In agreement with our hypothesis, mutations of the active site Asp abolish DFPase, PON1, and SMP30 OPase activity. Even though DFPase and SMP30 lose all lactonase activity with the Asp mutation, both PON1 and Drp35 retain minimal activity toward aromatic lactones. Additionally, crystallographic analyses are also under way; strong crystal leads from the Drp35 active site mutants and diffraction patterns of the DFPase active site Asp mutant have been obtained; subsequent structure solution from the latter is in progress. In the future, the mechanism and active site of these enzymes will be interrogated using inhibitor binding and subsequent structural analyses.
Keywords: mechanism, comparative enzymology, structural biology