The specific aims of this research are to discover and characterize new enzymes of the Nudix hydrolase superfamily and the aromatic phosphatase family of the HAD (Haloacid Dehalogenase) superfamily. The Nudix hydrolases are defined as enzymes that hydrolyze substrates consisting of a nucleoside diphosphate linked to some moiety x, and contain the signature sequence GX5EX7REUXEEXGU (U=l, L, or V). Nudix substrates include nucleoside triphosphates, nucleotide coenzymes, nucleotide sugars, diadenosine polyphosphates, and CDP-alcohols, and the Nudix hydrolases appear to perform vital cellular functions including removal of toxic compounds and modulation of natural metabolites to regulate signaling or biochemical pathways. We will focus on the Nudix hydrolases from Mycobacterium tuberculosis and Mycobacterium leprae, as potential novel antibiotic targets. The HAD superfamily is diverse, consisting of haloacid dehalogenases, phosphonatases, phosphomutases, and a number of distinctive phosphatase families. We will focus on a little defined phosphatase family within this superfamily, the aromatic phosphatases, formerly known as the nitrophenyl phosphatase family. Because only five enzymes with three activities have been identified for this family to date, the opportunity for discovery of new enzymatic activities is high. Concentrating on enzymes within operons or with known phenotypes will increase our success rate. Members of these two families will be cloned, expressed, purified, and characterized. Characterization will include biochemical analysis to determine substrate specificity and enzymatic activity, knockout mutagenesis with phenotypic and complementation studies to identify cellular function, and structural determination to understand structure-function relationships and for future drug design. Many of the enzymes discovered thus far appear to have important biological functions, thus making them good candidates as potential novel antibiotic targets in pathogenic bacteria. ? ? ?
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