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.

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

“…This may serve to compensate for the lack of a partially negative 2 0 -phosphate in NADP(H). 23,32,34,81 Of the mutants experimentally tested, only CbXR-RTT showed activity toward NADH and also increased activity for NADPH. This is consistent with the computational results in that the binding affinity for both cofactors was increased for this mutant (Table IV).…”

“…The residues with a higher net negative charge change in the NAD(H)-preferring enzymes, specifically the Asp and Glu residues, are thought to provide a significant portion of substrate specificity for NAD(H) by hydrogen bonding to one or both of the 2 0 -and 3 0 -OH and to compensate for the lack of a partially negative 2 0 -phosphate present in NADP(H). 23,32,34,81 Also, in three of the top five designs, position 272 was mutated to glutamic acid, indicating that this may be a critical mutation in changing the cofactor specificity of this enzyme.…”

“…Table I summarizes the best identified mutations involved in changing cofactor specificity (extending an earlier compilation). 22 Key successful redesigns include the work of Woodyer et al 23 that succeeded in Table I. Summary a of NAD(P)(H) Cofactor Engineering Studies Extending from Marohnic et al 22 Source relaxing the cofactor specificity of Pseudomonas stutzeri phosphite dehydrogenase from 100-fold in favor of nicotinamide adenine dinucleotide (NAD þ ) to threefold in favor of nicotinamide adenine dinucleotide phosphate (NADP þ ) using homology modeling and site-directed mutagenesis to identify and construct a double mutant.…”

“…This double mutant showed potential as an efficient in vitro NAD(P)(H) regeneration system for reductive biocatalysis. 23 Watanabe et al 24 used site-directed mutagenesis to change cofactor specificity of a Pichia stipitis NAD þ -dependent xylitol dehydrogenase (PsXDH) from NAD þ to NADP þ as part of an efficient biomass-ethanol conversion system. Their designs yielded greater activity for NADP þ than NAD þ after redesign.…”

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

“…Recombinant His-tagged phosphite dehydrogenase (6xHis-PtxD) was purified as described [21] with some modifications. After harvesting Escherichia coli from 400 ml LB medium after induction overnight at 25 °C the pellet was resuspended in 10 ml 50 mM MOPS 1 , pH 7.3; 200 mM NaCl; 10 mM -mercaptoethanol; 30 mM imidazole; 15% glycerol; 0.1 mM PMSF and cells lysed by ultrasonication on ice.…”