Mechanisms and Function in HAD Phosphotransferases

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The haloalkanoate dehalogenase superfamily (HADSF)is one of the largest and most ubiquitous enzyme families, with over 3,000 members in organisms ranging from bacteria to humans. The proposed studies will define, using steady-state and transient-state kinetics, X-ray structure determination, and bioinformatics, the mechanisms of catalysis and substrate recognition in selected HADSF phosphotransferases of biomedical importance. InAim 1the mechanism of catalysis of phosphoryl transfer in beta-phosphoglucomutase will be defined by measuring rate constants for individual steps of the catalytic cycle, identifying residues that stabilize the phosphorane intermediate using wild type and site- directed mutants, determining the mechanism of synchronizing cap closure and acid/base catalysis, and testing the role of cap domain closure in phosphorane formation. In part2 of Aim 1the structure of human alpha-phosphomannomutase will be determined and the roles of individual residues in substrate recognition, enzyme phosphorylation, substrate reorientation, conformational changes, and transition-state stabilization will befound for the wild-type enzyme and mutants clinically correlated with congenital disorders of glycosylation. Aim 2 targets the mechanisms of substrate recognition in two of the three HADSF subfamilies. In part 1, we will provide functional assignment to orphaned structures from the PDB corresponding to HADSF type MB phosphatases using a solvent cage method to identify leads for substrate screening. The apparent functional redundancy in sugar phosphatases within a single species will be probed by determining substrate range in homologs. In part 2, the Type III subfamily bacterial enzymes N- acyl-neuraminate-9-phosphate phosphatase and 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate phosphatase will be characterized structurally and in terms of substrate promiscuity in order to define the substrate specificity determinants and find if acid/base catalysis and substrate induced fit are operative.