Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase

John E. Hung, Emily J. Fogle, Harry D. Christman, Tyler W. Johannes, Huimin Zhao, William W. Metcalf, Wilfred A. Van Der Donk

Research output: Contribution to journalArticlepeer-review

Abstract

Phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri catalyzes the nicotinamide adenine dinucleotide-dependent oxidation of phosphite to phosphate. The enzyme belongs to the family of d-hydroxy acid dehydrogenases (DHDHs). A search of the protein databases uncovered many additional putative phosphite dehydrogenases. The genes encoding four diverse candidates were cloned and expressed, and the enzymes were purified and characterized. All oxidized phosphite to phosphate and had similar kinetic parameters despite a low level of pairwise sequence identity (39-72%). A recent crystal structure identified Arg301 as a residue in the active site that has not been investigated previously. Arg301 is fully conserved in the enzymes shown here to be PTDHs, but the residue is not conserved in other DHDHs. Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ∼100-fold decrease in kcat and an almost 700-fold increase in Km,phosphite for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher kcat than the parent PTDH, and a more modest increase in Km for phosphite (nearly 40-fold). Given these results, Arg301 may be involved in the binding and orientation of the phosphite substrate and/or play a catalytic role via electrostatic interactions. Three other residues in the active site region that are conserved in the PTDH orthologs but not DHDHs were identified (Trp134, Tyr139, and Ser295). The importance of these residues was also investigated by site-directed mutagenesis. All of the mutants had kcat values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

Original languageEnglish (US)
Pages (from-to)4254-4262
Number of pages9
JournalBiochemistry
Volume51
Issue number21
DOIs
StatePublished - May 29 2012

ASJC Scopus subject areas

  • Biochemistry

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