Understanding, optimization, and application of phosphite dehydrogenase: Advancing NAD(P)H regeneration

This dissertation describes the in depth characterization of wild-type (WT) phosphite dehydrogenase (PTDH), and its optimization for industrial NAD(P)H regeneration. PTDH catalyzes a unique chemical reaction, the NAD-dependent oxidation of phosphite to phosphate, but has significant homology with the D-hydroxyacid dehydrogenase family. However, no other D-hydroxyacid dehydrogenase tested could catalyze oxidation of phosphite to phosphate. A structural model was created based on homology to D-hydroxyacid dehydrogenases and utilized in combination with site-directed mutagenesis to study the roles of active site residues. Lys76 is a phosphite-binding residue that is unique among this protein family. Arg237, conserved throughout the family, is also involved in phosphite binding, whereas Glu266 is important for substrate orientation but not catalysis. His292 is essential and is likely the general base that deprotonates the water nucleophile. Primary kinetic isotope studies with WT and mutants suggest that the observed isotope effect (∼2.1) is the maximum intrinsic isotope effect and is the rate-limiting step of the catalyzed reaction. The pH-rate profile of the enzyme in combination with the mutagenesis studies suggests that a reverse protonation mechanism is a possibility for phosphite dehydrogenase.; NAD(P)H regeneration is an industrially important process that supports biocatalytic reactions that utilize these cofactors. PTDH shows promise for NAD(P)H regeneration and was therefore optimized for industrial application. The cofactor specificity of PTDH was relaxed by rational design using the homology model. Cofactor specificity was changed from 100-fold in favor of NAD to 3-fold in favor of NADP, with improvements in the catalytic efficiency (1000-fold for NADP). Utilizing directed evolution, the activity, expression, and thermostability of PTDH were also improved. A 3-fold increase in heterologous expression, 2-fold improvement in kcat, and 7000-fold increase in thermostability were achieved. The best mutants from each type of improvement were characterized in detail. Combined mutants with relaxed cofactor specificity, improved activity, stability, and expression level were tested in small-scale regeneration reactions for the production of several industrially interesting compounds such as xylitol and L- tert-leucine. The results obtained show that the combined mutant PTDH enzymes allow high productivity and conversion, representing one of the best NAD(P)H regeneration methods currently available.