9733552 Hasson This project focuses on the study of the structure and mechanism of benzoylformate decarboxylase (BFD), in the context of the homologous thiamin diphosphate (TDP)-dependent enzyme family. This family has salient features that predispose it to become a paradigm for comparative structural enzymology. The overall architecture of BFD closely resembles that of other family members, and cofactor and metal-binding residues are extremely well conserved. Surprisingly, none of the active-site residues that are not directly bound to cofactor are conserved, even between BFD and pyruvate decarboxylase. The results of this comparison represents a challenge to the usual expectations and understanding of enzyme evolution, and suggests that cofactor chemistry and the nature of the reaction intermediates have been dominant forces in the evolution of TDP-dependent enzymes. To appreciate the full implications of these preliminary studies, the mechanisms of BFD and related enzymes must be understood in detail. The goals of this project will be attained through a combination of X-ray crystallographic structure determination, site-directed mutagenesis, kinetic analysis, and protein chemistry. The objectives are (i) to determine the roles of various active-site residues of BFD; (ii) to probe the catalytic mechanism through the characterization of inhibitors; (iii) to understand the assembly of the mandelate pathway in a multienzyme complex; and (iv) to develop a hands-on computer laboratory course for undergraduates that can be implemented widely at other institutions, so that a generation of students can be given an appreciation of the fascination of structural enzymology and the tools to use publicly available structural information to inform their future careers. The beauty and power of an enzyme lies in the ability of the structure of its polypeptide chain to perform a particular function. Appreciation of these qualities in an enzyme must be accompanied by an understanding of how struct ure and function relate. Such an understanding can often be augmented by study of how enzymes have varied during evolution. This proposal aims to cultivate a basic understanding of enzyme structure in undergraduates, and to perform a study of a specific enzyme (benzoylformate decarboxylase) in the context of the family of enzymes to which it belongs, in order to increase understanding of cofactor-dependent catalysis and evolution of catalytic activity.
