Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.
Department of Biochemistry, Nicolaus Copernicus University, Torun, Lwowska 1, Poland.
Plant Physiol Biochem. 2018 Apr;125:126-135. doi: 10.1016/j.plaphy.2018.02.007. Epub 2018 Feb 9.
Indole-3-acetic acid (IAA) conjugation is one of mechanisms responsible for regulation of free auxin levels in plants. A new member of the serine carboxypeptidase-like (SCPL) acyltransferases family from Oryza sativa has been cloned and characterized. 1-O-indole-3-acetyl-β-D-glucose (1-O-IAGlc): myo-inositol acyltransferase (IAInos synthase) is an enzyme of IAA ester conjugates biosynthesis pathway that catalyzes transfer of IAA moiety from 1-O-IAGlc to myo-inositol forming IA-myo-inositol (IAInos). The OsIAA-At cDNA has been cloned and expressed using yeast and bacterial expression systems. Proteins produced in Saccharomyces cerevisiae and Escherichia coli contained 483 and 517 amino acids, respectively. The enzyme functionally expressed in both expression systems exhibits 1-O-IAGlc-dependent acyltransferase activity. Analysis of amino acid sequence confirmed that rice IAInos synthase belongs to the SCPL protein family. Recombinant IAInos synthases produced in yeast and bacterial expression systems have been partially characterized and their properties have been compared to those of the native enzyme obtained from 6-days-old rice seedlings by biochemical approach. The oligosaccharide component of the protein enzyme is not necessary for its catalytic activity. The native enzyme showed the lowest specific activity of 5.01 nmol min mg protein, whereas the recombinant enzymes produced in yeast and bacteria showed specific activity of 18.75 nmol min mg protein and 18.09 nmol min mg protein, respectively. The K values for myo-inositol were similar for all three forms of the enzyme: 1.38, 0.83, 1.0 mM for native, bacterial and yeast protein, respectively. Both recombinant forms of IAInos synthase and the native enzyme also have the same optimal pH of 7.4 and all of them are inhibited by phenylmethylsulfonyl fluoride (PMSF), specific inhibitor of serine carboxypeptidases.
吲哚-3-乙酸（IAA）的共轭是调节植物中游离生长素水平的机制之一。从水稻中克隆并鉴定了丝氨酸羧肽酶样（SCPL）酰基转移酶家族的一个新成员。1-O-吲哚-3-乙酰-β-D-葡萄糖（1-O-IAGlc）：肌醇酰基转移酶（IAInos 合酶）是 IAA 酯共轭生物合成途径的一种酶，它催化从 1-O-IAGlc 到肌醇的 IAA 部分转移，形成 IA-肌醇（IAInos）。利用酵母和细菌表达系统克隆并表达了 OsIAA-At cDNA。在酿酒酵母和大肠杆菌中产生的蛋白质分别含有 483 和 517 个氨基酸。在这两种表达系统中功能表达的酶表现出 1-O-IAGlc 依赖性酰基转移酶活性。氨基酸序列分析证实，水稻 IAInos 合酶属于 SCPL 蛋白家族。通过生化方法从 6 天大的水稻幼苗中获得的天然酶，对酵母和细菌表达系统中产生的重组 IAInos 合酶进行了部分表征，并比较了它们的性质。蛋白酶的寡糖成分对其催化活性不是必需的。天然酶的比活性最低，为 5.01 nmol min mg 蛋白，而酵母和细菌中产生的重组酶的比活性分别为 18.75 nmol min mg 蛋白和 18.09 nmol min mg 蛋白。对于所有三种形式的酶，肌醇的 K 值都相似：天然、细菌和酵母蛋白的 K 值分别为 1.38、0.83 和 1.0 mM。IAInos 合酶的两种重组形式和天然酶的最适 pH 值也相同，均为 7.4，并且它们都受到苯甲基磺酰氟（PMSF）的抑制，PMSF 是丝氨酸羧肽酶的特异性抑制剂。
