Improving Activity and Stability of Candida boidinii Formate Dehydrogenase Through Rational Active Site Engineering

ABSTRACT: Formate dehydrogenases represent a highly attractive tool for nicotinamide cofactor regeneration for NADH-dependent oxidoreductases which can drive biocatalytic reductions and oxygenations with formate as cheap, small, stoichiometric reductant. However, in the synthetic context, native FDHs are far less popular than other recycling systems despite clear benefits when it comes to atom economy, mainly due to the intrinsic, low specific activity of FDHs. While the active site architecture is generally highly conserved across many FDHs from very diverse origins, smaller patterns of dissonance in the active site's vicinity can serve as hotspots for the rational design of more effective variants. In this study, we have created a series of Candida boidinii FDH variants incorporating non-consensus amino acids found in Saccharomyces cerevisiae FDH. A double mutant (C23S/P285D) was identified that shows a significantly higher specific activity, an overall more desirable pH and temperature profile, and an improved stability profile.