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Title:Mechanistic studies to determine the catalytic roles of active site residues in phosphite dehydrogenase
Author(s):Hung, John
Director of Research:van der Donk, Wilfred A.
Doctoral Committee Chair(s):van der Donk, Wilfred A.
Doctoral Committee Member(s):Gerlt, John A.; Metcalf, William W.; Nair, Satish K.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Phosphite Dehydrogenase
enzymology
chemical biology
enzymes
enzyme kinetics
enzyme inhibition
Abstract:Phosphite dehydrogenase (PTDH) catalyzes the oxidation of phosphite to phosphate with the concurrent reduction of NAD+ to NADH. The mechanism of the reaction resembles a phosphoryl transfer reaction. A nucleophilic displacement reaction occurs on the phosphoryl group, with water or hydroxide attacking the phosphorus atom and hydride acting as the leaving group. Given the inherently poor nature of the hydride leaving group, PTDH presents a case of an enzyme catalyzing unusual chemistry. Although PTDH activity was initially believed to be unique to the enzyme from Pseudomonas stutzeri, searches of the protein databases have uncovered genes with high sequence identity to the original PTDH. When diverse members of this family were expressed and the purified proteins characterized, it was revealed that several orthologs of PTDH were able to catalyze the PTDH reaction with similar catalytic parameters. Sequence alignments of the PTDH orthologs showed that numerous enzyme residues are conserved amongst the PTDH family. In conjunction with the recently solved X-ray crystal structure of PTDH, several conserved residues that are present in the active site were identified and studied by mutagenesis experiments. Arg301 is conserved among known PTDHs and is in good position in the active site to act as the catalytic base in the reaction. Arg301 is important for efficient catalysis, as the Arg301Ala mutant displayed an approximately 100-fold decrease in kcat, and an approximately 700-fold increase in Km,phosphite. Chemical rescue and pH dependence experiments suggested that Arg301 acts as a positively charged residue in electrostatic activation of the reaction, rather than acting as the base. In addition, inhibition experiments indicated that binding of the sulfite competitive inhibitor in mutants of Arg301 was greatly decreased, suggesting that Arg301 plays an important role in substrate binding. The crystal structures of the Arg301Ala and Arg301Lys mutants of PTDH have been solved, and they are consistent with these proposed roles. In proteins, methionine residues are most commonly associated with hydrophobic interactions and steric effects. However, in the crystal structure of PTDH ternary complex, the sulfur of the conserved Met53 appears to interact with an oxygen atom on the sulfite competitive inhibitor. Mutagenesis experiments showed that Met53 is important for catalysis; mutations of this residue resulted in significantly decreased kcat, without changing Km for phosphite. A computational quantum mechanics/molecular mechanics (QM/MM) model was developed by the Mulholland Laboratory (University of Bristol, UK), which proposed that the side chain of Met53 stabilizes the transition state for hydride transfer during the PTDH reaction through an nπ* interaction between the Met53 sulfur and the His292 imidazolium. This hypothesis was supported by the experimental data, which indicated that mutation of Met53 affects the rate-limiting hydride transfer step in the reaction. In addition, a library of PTDH mutants was generated in an attempt to generate a PTDH capable of accepting thiophosphite as an alternative substrate through directed evolution. Several preliminary hits in this assay are currently being investigated. Successful mutation could allow for future analysis of the stereochemistry of the PTDH reaction. Attempts have also been made to determine if PTDH uses a covalent catalysis mechanism during phosphoryl transfer by detecting a phosphohistidine adduct using mass spectrometry.
Issue Date:2014-01-16
URI:http://hdl.handle.net/2142/46929
Rights Information:Copyright 2013 John Hung. Reproduced in part with permission from: Hung, J. E., Fogle, E. J., Christman, H. D., Johannes, T. W., Zhao, H. M., Metcalf, W. W., and van der Donk, W. A. (2012) Investigation of the Role of Arg301 Identified in the X-ray Structure of Phosphite Dehydrogenase, Biochemistry 51, 4254-4262. Copyright 2012 American Chemical Society.
Date Available in IDEALS:2014-01-16
2016-01-16
Date Deposited:2013-12


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