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首页> 外文期刊>Journal of Experimental Botany >Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification
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Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification

机译：质体定位拟南芥乙醛酸还原酶同工型的鉴定与表征：与胞质同工型的比较及其对细胞氧化还原稳态和醛解毒的影响

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摘要

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (Km glyoxylate=34 μM), and SSA to γ-hydroxybutyrate (Km SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (kcat/Km). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.
机译：将醛化学基团（即H-C = O）还原为相应醇的酶对于维持植物健康至关重要。最近，重组表达来自拟南芥（L.）Heynh（称为乙醛酸还原酶1或AtGR1）的胞质酶显示它有效催化乙醛酸和琥珀酸半醛（SSA）的体外还原。在本文中，基于网络的生物信息学工具揭示了第二个推定的GR cDNA（GenBank登录号AAP42747；在此指定为AtGR2），与GR1的氨基酸同源性为57％。从全长GR2 cDNA中删除编码假定的靶向信号的序列（N端43个氨基酸），并将所得的截短基因与分子伴侣GroES / EL在大肠杆菌中共表达，从而能够生产和纯化可溶性重组体蛋白。动力学分析表明，重组GR2催化乙醛酸转化为乙醇酸酯（K m 乙醛酸酯= 34μM），SSA转化为γ-羟基丁酸酯（K m SSA = 8.96 mM）。通过一种不可逆的，基于NADPH的机制。根据性能常数（k cat / K m ），GR2对乙醛酸酯的偏好比SSA高350倍。用与绿色荧光蛋白（GFP）连接的GR1瞬时转化的烟草（Nicotiana tabacum L.）悬浮细胞的荧光显微镜分析显示，GR1定位于胞质溶胶，而GR2-GFP通过其N中包含的靶向信息定位于质体-末端45个氨基酸。关于醛解毒和植物胁迫反应，讨论了不同的质体和胞质乙醛酸还原酶同工型的鉴定和表征。

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