The studies proposed in this new grant application will examine the structure and mechanism of action of the enzyme phosphonatase and extend structural and mechanistic studies to other members of the haloacid dehalogenase (HAD) enzyme superfamily. Each enzyme of this superfamily uses a conserved Asp residue to form either an acylphosphate-enzyme intermediate or an alkyl ester-enzyme intermediate. This chemistry is supported by a common structural scaffold. Phosphonatase catalyzes the hydrolysis of phosphonoacetaldehyde (P-Ald) to acetaldehyde and orthophosphate. In conjunction with 2-aminoethylphosphonate transaminase, phosphonatase functions in a two-step biodegradative pathway used to recycle P, N, and C from the ubiquitous natural phosphonate, 2-aminoethylphosphonate. Despite the wide range of known biological activities associated with natural and synthetic phosphonates, the enzymology of phosphonate metabolism is poorly characterized. The goal of these studies is to derive an understanding of the process of enzyme catalyzed C-P bond cleavage using phosphonatase as the model system. The first set of experiments proposed will test mechanistic models based on the recently determined phosphonatase X-ray structure (Allen laboratory) and on previous mechanistic studies (Dunaway-Mariano laboratory). Site-directed mutagenesis coupled with transient kinetic analysis will be used to test the chemical steps of the models. Crystallographic structure determinations carried out on dead-end complexes formed using substrate analogues, enzyme mutants, and chemically modified enzymes will be used to capture the structures of proposed reaction intermediates. The second set of experiments proposed will examine the active-site diversification of the HAD enzyme superfamily. The ability of phosphonatase to catalyze the reactions of other family members will be determined and protein engineering will be used to swap catalytic activities of two family members. To further probe the catalytic plasticity of the superfamily, the structure and mechanism of beta-phosphoglucomutase, a phosphotransferase from the HAD family will be examined. The goal of these studies is to derive an understanding of how the enzyme superfamily active site has been adapted to catalyze C-X, P-O and C-P bond cleavage in a variety of different substrate structures.

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National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Jones, Warren
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Boston University
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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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