Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, India; ICAR-Central Institute for Cotton Research, Nagpur, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, 247 667, India.
Insect Biochem Mol Biol. 2022 Aug;147:103812. doi: 10.1016/j.ibmb.2022.103812. Epub 2022 Jul 9.
Farnesol dehydrogenase (FDL) orchestrates the oxidation reaction catalyzing farnesol to farnesal, a key step in the juvenile hormone (JH) biosynthesis pathway of insects and hence, represents a lucrative target for developing insect growth regulators (IGRs). However, information on the structural and functional characterization of JH-specific farnesol dehydrogenase in insects remains elusive. Herein, we identified a transcript that encodes farnesol dehydrogenase (HaFDL) from Helicoverpa armigera, a major pest of cotton. The investigations of molecular assembly, biochemical analysis and spatio-temporal expression profiling showed that HaFDL exists as a soluble homo-tetrameric form, exhibits a broad substrate affinity and is involved in the JH-specific farnesol oxidation in H. armigera. Additionally, the study presents the first crystal structure of the HaFDL-NADP enzyme complex determined at 1.6 Å resolution. Structural analysis revealed that HaFDL belongs to the NADP-specific cP2 subfamily of the classical short-chain dehydrogenase/reductase (SDR) family and exhibits typical structural features of those enzymes including the conserved nucleotide-binding Rossman-fold. The isothermal titration calorimetry (ITC) showed a high binding affinity (dissociation constant, Kd, 3.43 μM) of NADP to the enzyme. Comparative structural analysis showed a distinct substrate-binding pocket (SBP) loop with a spacious and hydrophobic substrate-binding pocket in HaFDL, consistent with the biochemically observed promiscuous substrate specificity. Finally, based on the crystal structure, substrate modeling and structural comparison with homologs, a two-step reaction mechanism is proposed. Overall, the findings significantly impact and contribute to our understanding of farnesol dehydrogenase functional properties in JH biosynthesis in H. armigera.
法呢醇脱氢酶（FDL）催化法呢醇氧化为法呢醛，这是昆虫保幼激素（JH）生物合成途径中的关键步骤，因此，它是开发昆虫生长调节剂（IGR）的有吸引力的目标。然而，昆虫中 JH 特异性法呢醇脱氢酶的结构和功能特征的信息仍然难以捉摸。本文从棉铃虫（Helicoverpa armigera）中鉴定出一个编码法呢醇脱氢酶（HaFDL）的转录本，棉铃虫是棉花的主要害虫。分子组装、生化分析和时空表达谱的研究表明，HaFDL 以可溶性同源四聚体形式存在，具有广泛的底物亲和力，并参与 H. armigera 中 JH 特异性法呢醇的氧化。此外，该研究还首次报道了 HaFDL-NADP 酶复合物的晶体结构，分辨率为 1.6 Å。结构分析表明，HaFDL 属于 NADP 特异性 cP2 亚家族的经典短链脱氢酶/还原酶（SDR）家族，具有这些酶的典型结构特征，包括保守的核苷酸结合 Rossman 折叠。等温滴定量热法（ITC）显示 NADP 与酶的高结合亲和力（解离常数，Kd，3.43 μM）。比较结构分析显示 HaFDL 中存在独特的底物结合口袋（SBP）环和宽敞的疏水性底物结合口袋，与生化观察到的混杂底物特异性一致。最后，基于晶体结构、底物建模和与同源物的结构比较，提出了两步反应机制。总体而言，这些发现对理解 HaFDL 在 H. armigera JH 生物合成中的功能特性具有重要意义。
