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 (k /K ) /(k /K ) 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.
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