Departamento de Botánica, Ecología y Fisiología Vegetal, Instituto Andaluz de Biotecnología, Campus de Rabanales, Edif. C-4, 3a Planta, Universidad de Córdoba, 14071 Córdoba, Spain.
Physiol Plant. 2011 Dec;143(4):316-28. doi: 10.1111/j.1399-3054.2011.01507.x. Epub 2011 Oct 3.
γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is a very important enzyme in tocopherol biosynthesis in all photosynthetic organisms. In this paper, we present the functional characterization and expression analysis of γ-TMT from the unicellular green alga Chlamydomonas reinhardtii. Recombinant TMT1 enzyme was purified and characterized. The size of TMT1 subunit was estimated as 37 kDa by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), in accordance with the predicted molecular size after TMT1 cDNA sequence. Recombinant TMT1 also showed an apparent molecular mass of 37 kDa in its native conformation, suggesting that native TMT1 has a monomeric structure similar to the plant TMTs already characterized. pH and temperature dependence of TMT1 activity were also similar to plant TMTs. Substrate specificity studies showed that Chlamydomonas TMT1 is responsible for the conversion of γ- and δ-tocopherol to α- and β-tocopherol, respectively. The kinetic properties of Chlamydomonas recombinant γ-TMT activity were studied and γ-TMT1 has a similar affinity for γ- and δ-tocopherol. Promoter sequence analysis and expression analysis by northern blot revealed that tmt1 expression is strongly upregulated by high light and downregulated by low temperature. This regulatory pattern of tmt1 expression supports the idea that γ- and α-tocopherol play specific roles in the adaptation to growth under low temperature and high light stress conditions.
γ-生育酚甲基转移酶（γ-TMT）（EC 2.1.1.95）是所有光合生物生育酚生物合成中非常重要的酶。在本文中，我们介绍了单细胞绿藻莱茵衣藻 γ-TMT 的功能表征和表达分析。重组 TMT1 酶被纯化并进行了表征。TMT1 亚基的大小通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）估计为 37 kDa，与 TMT1 cDNA 序列预测的分子大小一致。重组 TMT1 在其天然构象中也表现出明显的 37 kDa 分子量，表明天然 TMT1 具有类似于已表征的植物 TMT 的单体结构。TMT1 活性的 pH 和温度依赖性也与植物 TMT 相似。底物特异性研究表明，衣藻 TMT1 负责将 γ-和 δ-生育酚分别转化为 α-和 β-生育酚。对衣藻重组 γ-TMT 活性的动力学特性进行了研究，发现 γ-TMT1 对 γ-和 δ-生育酚具有相似的亲和力。启动子序列分析和 northern blot 表达分析表明，tmt1 表达受高光强烈上调，受低温下调。tmt1 表达的这种调控模式支持 γ-和 α-生育酚在适应低温和高光胁迫条件下生长中发挥特定作用的观点。
