Biotin Synthesis in Plants. The First Committed Step of the Pathway Is Catalyzed by a Cytosolic 7-Keto-8-Aminopelargonic Acid Synthase
Biotin Synthesis in Plants. The First Committed Step of the Pathway Is Catalyzed by a Cytosolic 7-Keto-8-Aminopelargonic Acid Synthase
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植物中生物素的合成。
DOI：
10.1104/pp.105.070144
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发表时间：
2005
期刊：
影响因子：
7.4
通讯作者：
C. Alban
中科院分区：
文献类型：
--
作者：
Violaine Pinon;S. Ravanel;R. Douce;C. Alban
植物中生物素生物合成途径的生化和分子表征主要涉及生物素合酶。这种催化该途径最后一步的酶位于线粒体中。然而，该途径的其他酶在很大程度上是未知的。在这项研究中，基于基因组的方法使我们能够克隆拟南芥（Arabidopsis thaliana）编码7-酮-8-氨基壬酸（KAPA）合酶的cDNA，这是生物素合成途径的第一个关键酶，我们将其命名为AtbioF。该酶的功能通过对 KAPA 合酶反应缺陷的大肠杆菌突变体进行功能互补并通过测量体外活性来证明。重组 AtbioF 蛋白的过量生产和纯化能够全面表征酶的动力学特性，并对酶与其底物和产物的相互作用进行光谱研究。这是真核生物中 KAPA 合酶反应的首次表征。最后，绿色荧光蛋白靶向实验和蛋白质印迹分析均表明拟南芥 KAPA 合酶存在于细胞质中，从而揭示了植物生物素合成的独特区室，即细胞质和线粒体之间的分裂。还讨论了植物细胞中生物素合成和利用的复杂划分的意义及其在生物素代谢调节中的潜在重要性。
Biochemical and molecular characterization of the biotin biosynthetic pathway in plants has dealt primarily with biotin synthase. This enzyme catalyzing the last step of the pathway is localized in mitochondria. Other enzymes of the pathway are however largely unknown. In this study, a genomic-based approach allowed us to clone an Arabidopsis (Arabidopsis thaliana) cDNA coding 7-keto-8-aminopelargonic acid (KAPA) synthase, the first committed enzyme of the biotin synthesis pathway, which we named AtbioF. The function of the enzyme was demonstrated by functional complementation of an Escherichia coli mutant deficient in KAPA synthase reaction, and by measuring in vitro activity. Overproduction and purification of recombinant AtbioF protein enabled a thorough characterization of the kinetic properties of the enzyme and a spectroscopic study of the enzyme interaction with its substrates and product. This is the first characterization of a KAPA synthase reaction in eukaryotes. Finally, both green fluorescent protein-targeting experiments and western-blot analyses showed that the Arabidopsis KAPA synthase is present in cytosol, thus revealing a unique compartmentation of the plant biotin synthesis, split between cytosol and mitochondria. The significance of the complex compartmentation of biotin synthesis and utilization in the plant cell and its potential importance in the regulation of biotin metabolism are also discussed.
影响因子：
7.2
作者：
ABEL, S;THEOLOGIS, A
通讯作者：
THEOLOGIS, A