State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, PR China.
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Int J Biol Macromol. 2022 Jun 1;209(Pt B):1784-1791. doi: 10.1016/j.ijbiomac.2022.04.150. Epub 2022 Apr 30.
The biosynthesis of brasilane-type sesquiterpenoids (BTSs) attracts much attention owing to their unique skeleton of 5/6 bicyclic structure that contains five Me groups. Here, the crystal structures of a BTS cyclase TaTC6 from Trichoderma atroviride FKI-3849 and its complexes with farnesyl pyrophosphate (FPP) and analogue were reported. These structural information reveal that TaTC6 exploits a hydrophobic pocket to constrain the hydrocarbon region of FPP in a "U-shape" to facilitate the initial C1-C11 bond formation after pyrophosphate ionization. Following, four carbocations of reaction intermediates were molecularly docked into the hydrophobic pocket to reveal critical residues involved in the cyclization cascade. Finally, an S239-stabilized water molecule that is 3.9 Å away from the C8 of the last allyl cation may conduct hydration to quench the reaction cascade. Mutating S239 to alanine led to ca. 40% reduction in activity compared with the wild-type enzyme. The conservation of the residues that constitute the hydrophobic pocket is also discussed. Overall, this study will give an insight into the mechanism of how the active site of STCs constrain the conformation of the flexible FPP and series allylic carbocations for the complicated-ring formation and unusual carbon rearrangement in the biosynthesis of BTSs.
巴西烷型倍半萜（BTS）的生物合成因其独特的 5/6 双环骨架而备受关注，该骨架含有五个 Me 基团。本文报道了来自深绿木霉 FKI-3849 的 BTS 环化酶 TaTC6 的晶体结构及其与法呢基焦磷酸（FPP）和类似物的复合物。这些结构信息表明 TaTC6 利用疏水口袋将 FPP 的烃区域约束成“U 形”，以促进焦磷酸离子化后初始 C1-C11 键的形成。随后，将四个反应中间体的碳正离子分子对接入疏水口袋，以揭示参与环化级联的关键残基。最后，一个与最后一个烯丙基碳正离子距离为 3.9Å 的 S239 稳定的水分子可能进行水合作用来淬灭反应级联。将 S239 突变为丙氨酸会导致酶活性相对于野生型酶降低约 40%。还讨论了构成疏水口袋的残基的保守性。总的来说，这项研究将深入了解 STC 的活性位点如何约束柔性 FPP 的构象以及一系列烯丙基碳正离子，从而在 BTS 的生物合成中进行复杂环形成和不寻常的碳重排。
