NADP(H)-dependent biocatalysis without adding NADP(H).
NADP(H)-dependent biocatalysis without adding NADP(H).
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DOI：
10.1073/pnas.2214123120
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发表时间：
2023-01-03
影响因子：
11.1
通讯作者：
Armstrong, Fraser A.
中科院分区：
文献类型：
--
作者：
Herold, Ryan A.;Reinbold, Raphael;Schofield, Christopher J.;Armstrong, Fraser A.
在细胞内，催化多步过程（级联）的酶和辅因子通常被限制在一起-或者在外壳中（例如，细胞器）或通过物理缔合（代谢子）。纳米限制，为催化提供潜在的一般优势，也可以通过将酶及其可交换的辅因子装载到多孔的导电无机材料中来实现，从而使催化能够被引导，通电和电化学研究。这种纳米限制使得包含电活性铁氧还蛋白NADP+还原酶和异柠檬酸脱氢酶的级联能够在数天内具有活性，通过再循环异柠檬酸脱氢酶上携带的捕获的NADP（H）来催化大量异柠檬酸的彻底氧化。纳米限制极大地提高了辅因子依赖性级联催化的效率，并在概念上与生物进化有关，在生物进化中，复杂的有机分子可能在矿物的缝隙和裂缝中形成。异柠檬酸脱氢酶1（IDH 1）在静息状态下与其可交换辅因子NADP+/NADPH分子天然共纯化和结晶，NADP+/NADPH分子结合在同二聚体的每个单体中。我们报告了IDH 1的电化学研究，利用这一特性揭示了纳米限制的巨大优势，以提高多步酶催化级联反应的效率。当在纳米多孔导电氧化铟锡膜中与铁氧还蛋白NADP+还原酶共染色时，IDH 1仅使用与IDH 1共纯化并作为货物携带到电极孔中的NADP（H），将6 mM异柠檬酸盐（在4 mL中）完全电化学氧化为2-酮戊二酸盐（20 G）-该系统保持活性数天。现在可以通过循环伏安法定量的捕获的辅因子在该过程中经历约160，000次周转。从各种电催化实验的结果表明，当地的浓度的两个nanocolined酶位于周围的毫摩尔范围。拥挤和截留的组合导致NADP（H）氧化还原循环效率增加102至103倍。利用该方法在任一方向（氧化或还原）上驱动级联催化以及去除和替换底物的能力来研究野生型IDH 1与癌症相关R132 H变体之间辅因子结合的氧化还原状态依赖性差异，所述变体催化2 OG还原为2-羟基戊二酸而不是异柠檬酸氧化的“功能获得”。综合结果表明，纳米限制的力量，促进多步酶催化（在这种情况下通电和电化学验证），并揭示了烟酰胺辅因子作为氧化还原（氢化物）载体的动态作用的见解。
Within cells, enzymes and cofactors catalyzing multistep processes (cascades) are often confined together—either in enclosures (e.g., organelles) or via physical association (metabolons). Nanoconfinement, offering potential general advantages for catalysis, can also be achieved by loading enzymes and their exchangeable cofactors into a porous, electrically conducting inorganic material, thereby enabling catalysis to be channeled, energized, and investigated electrochemically. Such nanoconfinement enables a cascade comprising electroactive ferredoxin NADP+ reductase and isocitrate dehydrogenase to be active for days, catalyzing exhaustive oxidation of bulk isocitrate by recycling trapped NADP(H) carried in on isocitrate dehydrogenase. Nanoconfinement massively increases the efficiency of cofactor-dependent cascade catalysis and has conceptual relevance for prebiotic evolution where complex organic molecules might have formed in gaps and cracks of minerals. Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP+/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP+ reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo—the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 102 to 103-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the “gain of function” reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.
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DOI：
10.1021/acs.jpclett.1c01517
发表时间：
2021-07-08
期刊：
The journal of physical chemistry letters
影响因子：
--
作者：
Herold RA;Reinbold R;Megarity CF;Abboud MI;Schofield CJ;Armstrong FA
通讯作者：
Armstrong FA
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
12.9
作者：
Megarity, Clare F.;Weald, Thomas R. I.;Heath, Rachel S.;Turner, Nicholas J.;Armstrong, Fraser A.
通讯作者：
Armstrong, Fraser A.
影响因子：
4.8
作者：
Hektor, HJ;Kloosterman, H;Dijkhuizen, L
通讯作者：
Dijkhuizen, L
影响因子：
8
作者：
Monchietti, Paula;Lopez Rivero, Arleth S.;Catalano-Dupuy, Daniela L.
通讯作者：
Catalano-Dupuy, Daniela L.