Our primary research program is directed at a greater understanding of how enzymes are able to catalyze highly complex biochemical reactions with extraordinary rate enhancements and well-defined substrate profiles. We are interested in the identification of high-energy intermediates and transition states of enzyme-catalyzed reactions, and the role of specific residues residing in the active site that are responsible for binding and catalysis. These efforts have been enhanced to include the development of multi-disciplinary methods for the discovery of new enzymes that function to catalyze novel biochemical transformations. In addition, our laboratory is working to design novel approaches for the reconstruction of enzyme active sites in an effort to create new enzymes that are able to catalyze unprecedented biochemical transformations, including the detoxification of organophosphate nerve agents and chemo-enzymatic methods for the synthesis of antiviral therapeutics.
Capsular polysaccharides from Campylobacter jejuni
Campylobacer jejuni is the leading cause of food poisoning in the U.S. today. This organism synthesizes a complex capsular polysaccharide (CPS) on the surface of the bacterium that helps protect against the human immune response. In the most well-studied example, the CPS is encoded by a cluster of 35 genes for the biosynthesis of highly unusual carbohydrates and the machinery needed to assemble and decorate these carbohydrates into a unique polysaccharide.
J. P. Huddleston and F. M. Raushel, Functional characterization of Cj1427, a unique ping-pong dehydrogenase responsible for the oxidation of GDP-d-glycero-α-d-manno-heptose in campylobacter jejuni. Biochemistry 59, 1328–1337 (2020).
Z. W. Taylor, H. A. Brown, T. Narindoshvili , C. Q. Wenzel, C. M. Szymanski, H. M. Holden, F. M. Raushel, Discovery of a glutamine kinase required for the biosynthesis of the o-methyl phosphoramidate modifications found in the capsular polysaccharides of Ccampylobacter jejuni. J. Am. Chem. Soc. 139, 9463–9466 (2017).
Detoxification of organophosphate nerve agents
We are using directed evolution and rational design to create new enzymes with altered catalytic properties from existing protein templates. For this project we are making new enzymes for the catalytic destruction and detoxification of organophosphate nerve agents and designing novel enzyme catalysts for the enantioselective synthesis of value-added chiral synthons. These methods have been utilized for the construction of chiral precursors to potent antiviral prodrugs.
D. F. Xiang, A. N. Bigley, E. Desormeaux, T. Narindoshvili, F. M. Raushel, Enzyme-catalyzed kinetic resolution of chiral precursors to antiviral prodrugs. Biochemistry 58, 3204–3211 (2019).
A. N. Bigley, E. Desormeaux, D. F. Xiang, S. Y. Bae, S. P. Harvey, F. M. Raushel, Overcoming the challenges of enzyme evolution to adapt phosphotriesterase for v-agent decontamination. Biochemistry 58, 2039–2053 (2019).
Elucidation of enzyme reaction mechanisms
Our laboratory is committed to a greater understanding how enzymes work to catalyze biochemistry transformations with tremendous rate enhancements. We are using steady-state and transient state kinetic measurements to elucidate the structures of high energy reaction intermediates. These investigations are complimented by the synthesis of tight-binding inhibitors and the determination of the three dimensional enzyme structures by x-ray diffraction methods.
T. Hogancamp, M. Mabanglo, F. M. Raushel, Structure and reaction mechanism of the ligJ hydratase: an enzyme critical for the bacterial degradation of lignin in the protocatechuate 4,5-cleavage pathway. Biochemistry 57, 5841–5850 (2018).
A. N. Bigley, D. F. Xiang, T. Narindoshvili, C. Burgert, A. Hengge, F. M. Raushel, Transition state analysis of the reaction catalyzed by the phosphotriesterase from sphingobium TCM1. Biochemistry 58, 1246–1259 (2019).
To Academic Professional Track Faculty