Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration

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NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A/A 176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH-E175A/A 176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH-E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving DV values of 2.2 and 1.7, respectively, and DV/Km,phosphite values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH-E175A/A176R were 53 and 1.9 μM, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 μM, respectively. Using sulfite as a substrate mimic, the binding order was established, with the cofactor binding first and sulfite binding second. The low dissociation constant for the cofactor product NADPH combined with the reduced values for DV and Kcat implies that product release may become partially rate determining. However, product inhibition does not prevent efficient in situ NADPH regeneration by PTDH-E175A/A176R in a model system in which xylose was converted into xylitol by NADP-dependent xylose reductase. The in situ regeneration proceeded at a rate approximately fourfold faster with PTDH-E175A/A176R than with either WT PTDH or a NADP-specific Pseudomonas sp.101 formate dehydrogenase mutant with a total turnover number for NADPH of 2500.

Original languageEnglish (US)
Pages (from-to)3816-3827
Number of pages12
JournalFEBS Journal
Issue number15
StatePublished - Aug 2005


  • Biocatalysis
  • Cofactor regeneration
  • Dehydrogenases
  • Homology modelling
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry


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