NAD+激酶
酶动力学
辅因子
酶
甲酸脱氢酶
生物化学
蛋白质工程
生物
立体化学
化学
活动站点
作者
Liyuan Hu,Luyao Liu,Chunjun Zhan,Xiuxia Liu,Chunli Liu,Ye Li,Zhonghu Bai,Yankun Yang
标识
DOI:10.1002/cbic.202300587
摘要
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 Kph FDH, is a typical NAD + ‐specific FDH. However, investigations into engineering the cofactor specificity of Kph FDH have yet to be conducted. To develop an NADP + ‐specific variant of Kph FDH, we selected D195, Y196, and Q197 as mutation sites and generated twenty site‐directed variants. Through kinetic characterization, Kph FDH/V19 (D195Q/Y196R/Q197H) was identified as the variant with the highest specificity towards NADP + , with a ratio of catalytic efficiency ( k cat / K M ) NADP+ /( k cat / K M ) NAD+ of 129.226. Studies of enzymatic properties revealed that the optimal temperature and pH for the reduction reaction of NADP + catalyzed by Kph FDH/V19 were 45 °C and 7.5, respectively. The molecular dynamics (MD) simulation was performed to elucidate the mechanism of high catalytic activity of Kph FDH/V19 towards NADP + . Finally, Kph FDH/V19 was applied to an in vitro NADPH regeneration system with Meso ‐diaminopimelate dehydrogenase from Symbiobacterium thermophilum ( St DAPDH/H227V). This study successfully created a Kph FDH variant with high NADP + specificity and demonstrated its practical applicability in an in vitro NADPH regeneration system.
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