The broad, long-term objectives of this research are three-fold: 1) to discover and characterize new enzyme families to help properly annotate the growing body of data from genome sequencing projects, 2) to discover and characterize new enzymes that could be potential novel antibiotic targets, and 3) to better understand how enzymes recognize their substrates, which could lead to better drug design. Progress toward these objectives will be made by studying enzymes of two different enzyme families, the Nudix hydrolases that hydrolyze substrates composed of nucleoside diphosphate linked to some moiety """"""""x,"""""""" and a phosphatase family of the HAD (haloacid dehalogenase) superfamily. The Nudix hydrolases contain the signature sequence GX5EX7REUXEEXGU where U=I, L, or V, and appear to perform vital functions in the cell by removing toxic compounds or natural metabolites that would be harmful at elevated levels. The principal investigator will focus on the Nudix hydrolases from Mycobacterium tuberculosis, because they could be potential novel antibiotic targets due to their important cellular roles. As for the phosphatases of the HAD superfamily, the principal investigator will initially focus on the pyridoxal phosphatase YZGD from P. thiaminolyticus and NAGD from E. coli. She will clone the genes, and express, purify, and characterize enzymes of these two families. Characterization will include determining substrate specificity, pH optimum, metal ion requirements, kinetic values, and products released. The principal investigator will also continue structural determination of the enzymes through NMR spectroscopy or x-ray crystallography. Since all of the enzymes will be members of one of two distinct enzyme families, they will be related except for substrate specificity. Therefore, these two protein families make excellent model systems for asking the question, """"""""What determines substrate specificity?
| Hamm, Michelle L; McFadden, Emily J; Ghio, Michael et al. (2016) Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide. Bioorg Med Chem Lett 26:2014-7 |
| O'Handley, Suzanne F; Thirawatananond, Puchong; Kang, Lin-Woo et al. (2016) Kinetic and mutational studies of the adenosine diphosphate ribose hydrolase from Mycobacterium tuberculosis. J Bioenerg Biomembr 48:557-567 |
| Tirrell, Isaac M; Wall, Jennifer L; Daley, Christopher J et al. (2006) YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily. Biochem J 394:665-74 |
