Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.
Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.
复制标题
探测活性位点水在阿里斯托胆苷合酶催化的倍半萜环化反应中的作用。
DOI：
10.1021/acs.biochem.6b00343
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发表时间：
2016-05-24
期刊：
影响因子：
2.9
通讯作者：
Christianson DW
中科院分区：
文献类型：
--
作者：
Chen M;Chou WK;Al-Lami N;Faraldos JA;Allemann RK;Cane DE;Christianson DW
马兜铃烯合成酶（ATAS）是一种高保真萜类环化酶，可将法尼酯二磷酸转化为双环碳氢化合物马兜铃烯。先前确定的ATAS配合物的晶体结构揭示了可能与催化中间体相互作用的活性位点水分子：水分子“w”与S303和N299氢键，水分子“w1”和“w2”与Q151氢键，第四个水分子与Mg2+c离子配位。水在ATAS机制中没有明显的作用，因为酶只产生碳氢化合物产物。因此，这些水分子受到严格控制，因此它们不能与碳正离子中间体反应。8个ATAS突变体（S303A, S303H, S303D, N299A, N299L, N299A/S303A， Q151H和Q151E）的稳态动力学和产物分布分析表明，对催化作用的影响相对较小，但对倍半萜产物分布的影响显著。S303A、N299A、N299A/S303A和Q151H突变体的x射线晶体结构显示活性位点溶剂结构的扰动最小。8个突变体中有7个产生法尼醇和神经醇，可能是由于在最初形成的法尼基阳离子上加入了Mg2+c结合的水分子，但没有产生表明其他活性位点水分子活性增强的产物。然而，中间基因A在这些突变体中趋于积累。因此，除了mg2 +c结合水的可能反应性外，ATAS中的活性位点水分子并不直接参与催化化学，而是参与控制柔性底物和碳阳离子中间体构象的模板。
Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously-determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule is coordinated to the Mg2+c ion. There is no obvious role for water in the ATAS mechanism, since the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg2+c-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity ofMg2+c-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis, but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.