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学科主题生物工程
Assembling of Bioorthogonal Redox Systems Depending on an NAD Analog
Zhao ZB(赵宗保); Ji DB(纪德彬); Wang L(王磊); Liu WJ(刘武军); Wang JX(王金霞); Hou SH(侯淑华); Wang Q(王倩)
会议文集BITS 2nd Symposium on Enzymes and Biocatalysis-2011
会议名称BITS 2nd Symposium on Enzymes & Biocatalysis-2011
会议日期2011-4-25
2011
会议地点大连
页码61-0
出版者待补充
出版地待补充
合作性质分会口头报告
部门归属1816
主办者中国医药生物技术协会
英文摘要Biological redox chemistry is catalyzed by numerous enzymes depending largely on a few cofactors. Nicotinamide adenine dinucleotide (NAD) is one of the most important cofactors, is also involved in diverse non-redox processes. It remains challenging to disconnect one redox reaction from the other NAD-dependent cellular events. 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 multi-site saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME) from Escherichia coli, we identified the mutant ME-L3I0R/Q401C, activity of which was excellent with NFCD, but marginal with NAD. Moreover, wild type ME showed little activity with NFCD. Based on the insights abstracted from this process, we were able to generate a mutant of D-lactate dehydrogenase (DLDH) from Lacrobacillus helvettcus, DLDH-V152R, which was fully active with NFCD. Using the system contained ME-L31ORIQ401C and DLDH-V152R, we showed that the biotransformation of L-malate into D-Lactate could be done in the presence of a catalytic amount of NFCD, that is to say, NFCD was recyclable by two engineered enzyme. These bioorthogonal systems should be useful in terms of developing bioanalytical methods and producing biochemicals. Our approach opened the window to engineer bioorthogonal redox systems, which will provide us unique tools for systems biology and synthetic biology researches. We will discuss this in more detail during the conference.; Biological redox chemistry is catalyzed by numerous enzymes depending largely on a few cofactors. Nicotinamide adenine dinucleotide (NAD) is one of the most important cofactors, is also involved in diverse non-redox processes. It remains challenging to disconnect one redox reaction from the other NAD-dependent cellular events. 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 multi-site saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME) from Escherichia coli, we identified the mutant ME-L3I0R/Q401C, activity of which was excellent with NFCD, but marginal with NAD. Moreover, wild type ME showed little activity with NFCD. Based on the insights abstracted from this process, we were able to generate a mutant of D-lactate dehydrogenase (DLDH) from Lacrobacillus helvettcus, DLDH-V152R, which was fully active with NFCD. Using the system contained ME-L31ORIQ401C and DLDH-V152R, we showed that the biotransformation of L-malate into D-Lactate could be done in the presence of a catalytic amount of NFCD, that is to say, NFCD was recyclable by two engineered enzyme. These bioorthogonal systems should be useful in terms of developing bioanalytical methods and producing biochemicals. Our approach opened the window to engineer bioorthogonal redox systems, which will provide us unique tools for systems biology and synthetic biology researches. We will discuss this in more detail during the conference.
语种英语
文献类型会议论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/116040
专题中国科学院大连化学物理研究所
通讯作者Zhao ZB(赵宗保)
推荐引用方式
GB/T 7714
Zhao ZB,Ji DB,Wang L,et al. Assembling of Bioorthogonal Redox Systems Depending on an NAD Analog[C]. 待补充:待补充,2011:61-0.
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