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.
In Aim 1, the 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 part 2 of Aim 1, the 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 be found 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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061099-06A1
Application #
7033305
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Jones, Warren
Project Start
2000-04-01
Project End
2010-02-28
Budget Start
2006-03-10
Budget End
2007-02-28
Support Year
6
Fiscal Year
2006
Total Cost
$505,768
Indirect Cost
Name
Boston University
Department
Physiology
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Ji, Tianyang; Zhang, Chunchun; Zheng, Li et al. (2018) Structural Basis of the Molecular Switch between Phosphatase and Mutase Functions of Human Phosphomannomutase 1 under Ischemic Conditions. Biochemistry 57:3480-3492
Huang, Hua; Patskovsky, Yury; Toro, Rafael et al. (2011) Divergence of structure and function in the haloacid dehalogenase enzyme superfamily: Bacteroides thetaiotaomicron BT2127 is an inorganic pyrophosphatase. Biochemistry 50:8937-49
Wu, Rui; Garland, Megan; Dunaway-Mariano, Debra et al. (2011) Homo sapiens dullard protein phosphatase shows a preference for the insulin-dependent phosphorylation site of lipin1. Biochemistry 50:3045-7
Lu, Zhibing; Dunaway-Mariano, Debra; Allen, Karen N (2011) The X-ray crystallographic structure and specificity profile of HAD superfamily phosphohydrolase BT1666: comparison of paralogous functions in B. thetaiotaomicron. Proteins 79:3099-107
Wang, Liangbing; Huang, Hua; Nguyen, Henry H et al. (2010) Divergence of biochemical function in the HAD superfamily: D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB). Biochemistry 49:1072-81
Nguyen, Henry H; Wang, Liangbing; Huang, Hua et al. (2010) Structural determinants of substrate recognition in the HAD superfamily member D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) . Biochemistry 49:1082-92
Lu, Zhibing; Wang, Liangbing; Dunaway-Mariano, Debra et al. (2009) Structure-function analysis of 2-keto-3-deoxy-D-glycero-D-galactonononate-9-phosphate phosphatase defines specificity elements in type C0 haloalkanoate dehalogenase family members. J Biol Chem 284:1224-33
Dai, Jianying; Finci, Lorenzo; Zhang, Chunchun et al. (2009) Analysis of the structural determinants underlying discrimination between substrate and solvent in beta-phosphoglucomutase catalysis. Biochemistry 48:1984-95
Allen, Karen N; Dunaway-Mariano, Debra (2009) Markers of fitness in a successful enzyme superfamily. Curr Opin Struct Biol 19:658-65
Peisach, Ezra; Wang, Liangbing; Burroughs, A Maxwell et al. (2008) The X-ray crystallographic structure and activity analysis of a Pseudomonas-specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function. Proteins 70:197-207

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