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玉米C4型NADP-苹果酸酶。在大肠杆菌中的表达及推定核苷结合位点定点突变体的特性分析

Maize C4 NADP-malic enzyme. Expression in Escherichia coli and characterization of site-directed mutants at the putative nucleoside-binding sites.

作者信息

Detarsio Enrique, Wheeler Mariel C Gerrard, Campos Bermúdez Valeria A, Andreo Carlos S, Drincovich María F

机构信息

Centro de Estudios Fotosintéticos y Bioquimicos (CEFOBI), CONICET, Fund. M. Lillo, Universidad Nacional de Rosario, Suipacha 531, Argentina.

出版信息

J Biol Chem. 2003 Apr 18;278(16):13757-64. doi: 10.1074/jbc.M212530200. Epub 2003 Jan 31.

Abstract

Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO(2), and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C(4)-specific NADP-ME has evolved from C(3)-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C(4)-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and K(m) for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the k(cat) was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C(4) NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.

摘要

苹果酸酶在二价金属离子存在的情况下催化L-苹果酸的氧化脱羧反应,生成丙酮酸、二氧化碳和NAD(P)H。在植物中,NADP-苹果酸酶(NADP-ME)的不同同工型参与了广泛的代谢途径。C4植物特有的NADP-ME是从C3型苹果酸酶进化而来的,其独特的动力学和调节特性表明它是一种独特且专门化的NADP-ME形式。在本研究中,玉米成熟的C4植物特有的NADP-ME在大肠杆菌中表达。重组酶与先前表征的天然存在的NADP-ME相比,具有基本相同的物理化学性质和底物K(m)值。然而,催化常数(k(cat))几乎高出7倍,这可能表明先前从玉米叶片中纯化的酶部分无活性。重组NADP-ME还具有非常低的固有NAD依赖性活性。通过定点诱变构建了NADP-ME在假定的NADP结合位点(Gsite5V、Gsite2V、A392G、A387G和R237L)的五个突变体,并纯化至同质。通过动力学测量、圆二色性和固有荧光光谱推断这些残基在底物结合和/或催化反应中的参与情况。将获得的结果与基于相关动物NAD(P)-ME的晶体学研究预测的玉米C4 NADP-ME三维模型进行比较。本文提供的数据代表了植物NADP-ME的首次原核表达,并揭示了有关突变氨基酸在底物结合和/或催化中的参与情况的宝贵见解。

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