Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, India.
Department of Biochemistry, Central University of Haryana, Mahendergarh, 123031, Haryana, India.
Planta. 2021 Jan 4;253(1):20. doi: 10.1007/s00425-020-03514-y.
The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.
已经从印度楝（Neem）中克隆并鉴定了重组咖啡酸 3-O-甲基转移酶基因。该基因参与阿魏酸生物合成，阿魏酸是木质素生物合成的关键中间成分。印度楝是一种备受推崇的传统药用植物，其植物化学物质以其 limonoids 而闻名。除了 limonoids，酚类化合物也非常独特，这为 Neem 增添了更多的药用属性。咖啡酸是此类酚类化合物之一，它可以在适当的甲基转移酶的催化作用下，酶促转化为另一种生物活性植物分子阿魏酸。这种转化需要供体将一个甲基转移到咖啡酸上。在这项研究中，从 Neem（NCOMT）果实中分离并异源表达了咖啡酸 3-O-甲基转移酶基因。纯化了重组 NCOMT 酶，该酶显示出高效催化咖啡酸转化为阿魏酸的能力，阿魏酸是一种极具潜力的药物化合物。对纯化的重组酶进行了物理化学特性分析，以研究其催化作用。对 NCOMT 基因的组织广泛表达分析揭示了有趣的转录丰度模式，反映了其在果实组织发育中的作用。此外，还在印度麻和罗勒属植物中异源过表达了 NCOMT，以分析其在植物体内阿魏酸生物合成中的作用。因此，该研究为 NCOMT 在木质素生物合成途径中作为内源性阿魏酸生物合成酶提供了深入的见解，木质素是一种重要的结构成分。据我们所知，NCOMT 是从 Neem 中纯化和动力学表征的第二种代谢酶。这项研究也可能具有重要的应用前景，因为对 NCOMT 异源表达的观察表明，它参与了植物体内阿魏酸的体内池的维持。因此，涉及 NCOMT 的研究为在异源系统中生物合成潜在治疗重要的植物分子开辟了新的代谢工程方法维度。
