Institute of Systems Biology, Universiti Kebangsaan Malaysia, UKM Bangi, Bangi, Selangor, 43600, Malaysia.
Institute of Systems Biology, Universiti Kebangsaan Malaysia, UKM Bangi, Bangi, Selangor, 43600, Malaysia; Enzyme & Microbial Technology Center (EMTech), Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, UPM Serdang, Selangor, 43400, Malaysia.
Plant Physiol Biochem. 2021 Apr;161:143-155. doi: 10.1016/j.plaphy.2021.01.050. Epub 2021 Feb 5.
The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP as coenzyme. The substrate specificity studies showed trans, trans-farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP-dependent farnesol dehydrogenase. The docking of trans, trans-farnesol and NADP into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control.
植物中的保幼激素（JH）被认为是一种植物防御策略，特别是针对昆虫取食。法呢醇氧化为法呢酸是保幼激素生物合成途径中的关键步骤。本文介绍了可可树法呢醇脱氢酶的纯化和特性，该酶可催化法呢醇氧化为法呢醛。通过亲和层析将重组酶纯化至明显均一。从推导的氨基酸序列、系统发育、底物特异性、动力学参数、结构建模和对接模拟等方面对纯化酶进行了表征。系统发育分析表明，可可树法呢醇脱氢酶（TcFolDH）与拟南芥法呢醇脱氢酶基因密切相关。TcFolDH 单体具有较大的 N 端结构域，采用典型的 Rossmann 折叠结构，含有 GxxGxG 基序（NADP(H)结合域）和较小的 C 端结构域。该酶是由 36 kDa 亚基组成的三聚体。TcFolDH 对 NADP 作为辅酶具有高度特异性。底物特异性研究表明，反式，反式法呢醇是 TcFolDH 的最优选底物，表明纯化的酶是 NADP 依赖性法呢醇脱氢酶。将反式，反式法呢醇和 NADP 对接进入酶的活性位点，显示了重要的残基及其相互作用，这些残基和相互作用参与了 TcFolDH 的底物和辅酶结合。考虑到 JH 在昆虫和植物中广泛存在，深入了解 JH 生物合成途径中间酶的重组生产可能有助于为昆虫控制提供一种潜在的方法。
