Creating NADP⁺‐Specific Formate Dehydrogenases from Komagataella phaffii by Enzymatic Engineering

Abstract

Most natural formate dehydrogenases (FDHs) exhibit NAD+ specificity, making it imperative to explore the engineering of FDH cofactor specificity for NADPH regeneration systems. The endogenous FDH of Komagataella phaffii (K. phaffii), termed KphFDH, is a typical NAD+‐specific FDH. However, investigations into engineering the cofactor specificity of KphFDH have yet to be conducted. To develop an NADP+‐specific variant of KphFDH, we selected D195, Y196, and Q197 as mutation sites and generated twenty site‐directed variants. Through kinetic characterization, KphFDH/V19 (D195Q/Y196R/Q197H) was identified as the variant with the highest specificity towards NADP+, with a ratio of catalytic efficiency (kcat/KM)NADP+/(kcat/KM)NAD+ of 129.226. Studies of enzymatic properties revealed that the optimal temperature and pH for the reduction reaction of NADP+ catalyzed by KphFDH/V19 were 45°C and 7.5, respectively. The molecular dynamics (MD) simulation was performed to elucidate the mechanism of high catalytic activity of KphFDH/V19 towards NADP+. Finally, KphFDH/V19 was applied to an in vitro NADPH regeneration system with Meso‐diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH/H227V) . This study successfully created a KphFDH variant with high NADP+ specificity and demonstrated its practical applicability in an in vitro NADPH regeneration system.