Grant: Discovery Grants Program - Individual

My research program seeks to improve our understanding of enzymes, studying the relationship between structure, function, and catalysis in the context of enzymatic evolution. This program trains students at the interface of chemistry and biology. Our laboratory is a leading authority on the enzyme dihydrodipicolinate synthase (DHDPS), which catalyzes the first step in the biosynthesis of crucial components of bacterial cell walls and is thus an antibiotic target. DHDPS activity is inhibited by the binding of two molecules of lysine to an allosteric site. Our laboratory has shown that the inhibition results from interruption of the internal motions of DHDPS necessary for catalysis. Our lab has reported by far the most potent known inhibitor of DHDPS, and we have just developed a mutant of DHDPS from Campylobacter jejuni that can detect inhibitor binding by a change in fluorescence. We will adapt this system into a rapid method of screening candidate inhibitors. We are working with collaborators to determine specific effects of inhibitors on the dynamics of the protein using crystallography and computational methods. This will enable us to identify new lead compounds for antibiotics, and to help other scientists understand the principles of allosteric inhibition. DHDPS is part of a family of enzymes called type 1 aldolases. Examination of the similarities of DHDPS with another aldolase family member known as NahE led us to predict, correctly, that NahE could catalyze an aldol-transimination reaction, resulting in a novel quinoline-generating process we can carry out on gram scale (which is known as biocatalysis). We are now exploring more applications of this enzyme, including a catalytic reaction never observed before in a naturally occurring aldolase that provides access to high-value pharmaceuticals. We will work to understand the details of NahE catalysis so we can design mutants with enhanced substrate scope and stereoselectivity, and we will develop multi-enzyme processes to synthesize new products under mild and nontoxic conditions. Glucocerebrosidase hydrolyzes glucosyl ceramide, and malfunction of the human lysosomal version of this enzyme, known as GBA1, results in Gaucher's disease and is a biomarker for Parkinson's disease. Observing GBA1 activity in cells can be difficult due to the presence of similar enzymes, such as non-lysosomal GBA2, whose malfunction also leads to serious health conditions. Conduritol aziridines (CAZs) are sugar analogs that can bind to these enzymes selectively and inactivate them. Our laboratory has invented a new class of potent and selective CAZs. We will expand this technology to address a long-standing problem of differentiating GBA1 and GBA2 by selective inactivation. Our new CAZs will be useful for targeting and eliminating activity and give new insight into the active site architecture so that other scientist can understand these and related enzymes.