title: Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase.
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 k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher k(cat) than the parent PTDH, and a more modest increase in K(m) 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 k(cat) values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.
authors.affiliation: Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Hung
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Fogle
authors.forename: Harry D
authors.initials: HD
authors.lastname: Christman
authors.forename: Tyler W
authors.initials: TW
authors.lastname: Johannes
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.forename: William W
authors.initials: WW
authors.lastname: Metcalf
authors.forename: Wilfred A
authors.initials: WA
authors.lastname: van der Donk
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D044223:Pseudomonas stutzeri / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001120:Arginine; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase