title: Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design.
abstract: Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both separately and in combination resulted in PTDH mutants with relaxed cofactor specificity. All three mutants exhibited significantly better catalytic efficiency for both cofactors, with the best kinetic parameters displayed by the double mutant, which had a 3.6-fold higher catalytic efficiency for NAD(+) and a 1000-fold higher efficiency for NADP(+). The cofactor specificity was changed from 100-fold in favor of NAD(+) for the wild-type enzyme to 3-fold in favor of NADP(+) for the double mutant. Isoelectric focusing of the proteins in a nondenaturing gel showed that the replacement with more basic residues indeed changed the effective pI of the protein. HPLC analysis of the enzymatic products of the double mutant verified that the reaction proceeded to completion using either substrate and produced only the corresponding reduced cofactor and phosphate. Thermal inactivation studies showed that the double mutant was protected from thermal inactivation by both cofactors, while the wild-type enzyme was protected by only NAD(+). The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity. The rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Woodyer
authors.forename: Wilfred A
authors.initials: WA
authors.lastname: van der Donk
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D004795:Enzyme Stability / Q000235:genetics; D029968:Escherichia coli Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000737:chemistry; D009536:Niacinamide / Q000138:chemical synthesis* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D009536:Niacinamide; D018698:Glutamic Acid; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase; D000409:Alanine