Threonine-insensitive Homoserine Dehydrogenase from Soybean GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY
Threonine-insensitive Homoserine Dehydrogenase from Soybean GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY
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DOI：
10.1074/jbc.m109.068882
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发表时间：
2010-01-08
影响因子：
4.8
通讯作者：
Jez, Joseph M.
中科院分区：
文献类型：
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作者：
Schroeder, Amy C.;Zhu, Chuanmei;Jez, Joseph M.
天冬氨酸激酶（AK）和高丝氨酸脱氢酶（HSD）作为天冬氨酸氨基酸途径中分支点的关键调节酶起作用，并且被苏氨酸反馈抑制。在植物中，AK和双功能AK-HSD酶的生物化学特征已经被表征，但单功能HSD的分子特性尚未被研究。为了研究HSD的作用，我们从大豆中克隆了编码单功能HSD（GmHSD）的cDNA和基因。使用异源表达和纯化的GmHSD，初始速度和产物抑制研究支持一个有序的双双动力学机制，其中烟酰胺辅因子结合第一和叶最后的反应序列。GmHSD的苏氨酸抑制在高于生理水平1000倍以上的浓度（Ki = 160-240 mM）下发生。这与大豆中对苏氨酸抑制敏感的两种AK-HSD同种型（Ki类似于150 μ M）相反。此外，GmHSD不受其他谷氨酸衍生氨基酸的抑制。大豆组织中苏氨酸抗性与苏氨酸敏感的HSD活性的比率不同，可能反映了对氨基酸生物合成的不同需求。这是第一次克隆和详细的生化特性的单功能反馈不敏感的HSD从任何植物。苏氨酸抗性HSD提供了一种有用的生物技术工具，用于操纵天冬氨酸氨基酸途径以增加植物中苏氨酸和甲硫氨酸的产量，从而改善营养含量。
Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K-i = 160-240 mM) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K-i similar to 150 mu M). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.