Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis.
Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.
McLachlan
Michael J
MJ
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Johannes
Tyler W
TW
Zhao
Huimin
H
D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature / Q000009:adverse effects; D016296:Mutagenesis; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D013329:Structure-Activity Relationship
D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase