Creation of Bioorthogonal Redox Systems Depending on Nicotinamide Flucytosine Dinucleotide

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• 作者：Debin Ji ; Lei Wang ; Shuhua Hou ; Wujun Liu ; Jinxia Wang ; Qian Wang ; Zongbao K. Zhao
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：December 28, 2011
• 年：2011
• 卷：133
• 期：51
• 页码：20857-20862
• 全文大小：307K
• 年卷期：v.133,no.51(December 28, 2011)
• ISSN：1520-5126

文摘

Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.