Hirte Max, Meese Nicolas, Mertz Michael, Fuchs Monika, Brück Thomas B
Werner Siemens Chair of Synthetic Biotechnology, Department of Chemistry, Technical University of Munich, Munich, Germany.
Front Chem. 2018 Apr 10;6:101. doi: 10.3389/fchem.2018.00101. eCollection 2018.
Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of the enzyme's active site and that the geranylgeranyl diphosphate derived pyrophosphate moiety remains in the ACS active site thereby directing the cyclization process. Our cumulative data confirm that amino acids constituting the G-loop of diterpene synthases are involved in the open to the closed, catalytically active enzyme conformation. This study demonstrates that a simple and rapid biomolecular modeling procedure can predict catalytically relevant amino acids. The approach reduces computational and experimental screening efforts for diterpene synthase structure-function analyses.
二萜合酶催化复杂的多步碳-碳偶联反应，从而将通用的脂肪族前体香叶基香叶基二磷酸转化为多样的烯烃大环化合物，这些化合物构成了二萜天然产物家族结构多样性的基础。由于二萜合酶的催化相关晶体结构稀少，基于同源性的生物分子建模技术为研究该酶的反应机制提供了一条替代途径。然而，精确鉴定催化相关氨基酸具有挑战性，因为这些模型在进行底物对接研究之前需要仔细的制备和优化技术。在这类蛋白质中进行靶向氨基酸替换可能会引发以碳正离子为中心的反应级联的过早淬灭。对那些替代环化产物的结构表征有助于阐明环化反应级联，并为复杂的大环合成子提供新的来源。在本研究中，使用简化的生物分子建模策略获得了对真菌双功能蚜二萜-16-β-醇合酶结构和功能的新见解。所应用的优化方法能够快速生成可靠的蛋白质-配体复合物，从而准确鉴定催化相关氨基酸。在我们的建模数据指导下，对ACS进行突变导致鉴定出催化相关的ACS氨基酸网络I626、T657、Y658、A786、F789和Y923。此外，ACS氨基酸替换Y658L和D661A导致从顺式-柯巴基二磷酸到蚜二萜-16-β-醇的环化反应级联过早终止。这两个ACS突变体分别生成了二萜大环化合物顺式-柯巴醇和一种次要的、非羟基化的与半日花烷相关的二萜。我们的生物分子建模和突变研究表明，ACS底物环化发生在酶活性位点的一个空间受限位置，并且香叶基香叶基二磷酸衍生的焦磷酸部分保留在ACS活性位点，从而指导环化过程。我们的累积数据证实，构成二萜合酶G环的氨基酸参与了从开放到闭合的、具有催化活性的酶构象转变。本研究表明，一种简单快速的生物分子建模程序可以预测催化相关氨基酸。该方法减少了二萜合酶结构-功能分析的计算和实验筛选工作。
