Two Arabidopsis genes (IPMS1 and IPMS2) encode isopropylmalate synthase, the branchpoint step in the biosynthesis of leucine
Two Arabidopsis genes (IPMS1 and IPMS2) encode isopropylmalate synthase, the branchpoint step in the biosynthesis of leucine
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DOI：
10.1104/pp.106.085555
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发表时间：
2007-02-01
期刊：
影响因子：
7.4
通讯作者：
Gershenzon, Jonathan
中科院分区：
文献类型：
--
作者：
de Kraker, Jan-Willem;Luck, Katrin;Gershenzon, Jonathan
拟南芥 (Arabidopsis thaliana) IPMS1 (At1g18500) 和 IPMS2 (At1g74040) cDNA 在大肠杆菌中的异源表达产生异丙基苹果酸合酶 (IPMS；EC 2.3.3.13)。这些酶催化亮氨酸 (Leu) 生物合成的第一个专用步骤，即乙酰辅酶 A (CoA) 和 2-氧代异戊酸的羟醛型缩合，产生苹果酸异丙酯。 IPMS1 和 IPMS2 的大多数生化特性相似：最适 pH 范围约为 pH 8.5，Mg2+ 作为辅助因子，Leu 反馈抑制，2-氧代异戊酸的 K-m 约为 300 mu M，V-max 约为 2 x 10(3) mu mol min(-1) g(-1)。然而，IPMS1和IPMS2的乙酰辅酶A K-m（分别为45μM和16μM）和表观四级结构（分别为二聚体和四聚体）不同。 IPMS1 的敲除插入突变体显示缬氨酸含量增加，但亮氨酸含量没有变化； IPMS2 的两个插入突变体没有显示可溶性氨基酸含量的任何变化。显然，在植物中，每个基因都可以充分补偿另一个基因的缺失，这与现有的微阵列和逆转录聚合酶链式反应数据一致，这些数据表明这两个基因在所有发育阶段的所有器官中都表达。两种编码的蛋白质在体外接受长度从乙醛酸到2-氧代己酸的2-含氧酸底物，并以低速率催化乙酰辅酶A和4-甲硫基-2-氧代丁酸的缩合，即涉及硫代葡萄糖苷链延长的反应，通常由甲硫基烷基苹果酸合酶催化。鉴于 IPMS 和甲硫基烷基苹果酸合酶的氨基酸序列同一性 (60%) 和底物特异性的重叠，讨论了它们之间的进化关系。
Heterologous expression of the Arabidopsis (Arabidopsis thaliana) IPMS1 (At1g18500) and IPMS2 (At1g74040) cDNAs in Escherichia coli yields isopropylmalate synthases (IPMSs; EC 2.3.3.13). These enzymes catalyze the first dedicated step in leucine (Leu) biosynthesis, an aldol-type condensation of acetyl-coenzyme A (CoA) and 2-oxoisovalerate yielding isopropylmalate. Most biochemical properties of IPMS1 and IPMS2 are similar: broad pH optimum around pH 8.5, Mg2+ as cofactor, feedback inhibition by Leu, K-m for 2-oxoisovalerate of approximately 300 mu M, and a V-max of approximately 2 x 10(3) mu mol min(-1) g(-1). However, IPMS1 and IPMS2 differ in their K-m for acetyl-CoA (45 mu M and 16 mu M, respectively) and apparent quaternary structure (dimer and tetramer, respectively). A knockout insertion mutant for IPMS1 showed an increase in valine content but no changes in Leu content; two insertion mutants for IPMS2 did not show any changes in soluble amino acid content. Apparently, in planta each gene can adequately compensate for the absence of the other, consistent with available microarray and reverse transcription-polymerase chain reaction data that show that both genes are expressed in all organs at all developmental stages. Both encoded proteins accept 2-oxo acid substrates in vitro ranging in length from glyoxylate to 2-oxohexanoate, and catalyze at a low rate the condensation of acetyl-CoA and 4-methylthio-2-oxobutyrate, i.e. a reaction involved in glucosinolate chain elongation normally catalyzed by methylthioalkylmalate synthases. The evolutionary relationship between IPMS and methylthioalkylmalate synthase enzymes is discussed in view of their amino acid sequence identity (60%) and overlap in substrate specificity.