Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, United States.
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, United States.
Biochemistry. 2024 Nov 5;63(21):2904-2915. doi: 10.1021/acs.biochem.4c00547. Epub 2024 Oct 14.
In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from , with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1-C11 bond. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case, well fit by a curled-up tetraethylene glycol molecule recruited, presumably, from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotation of some active-site residues in comparison to the 2FFPP-structure. Most notably, W56 and F183 undergo 90° rotations between the 2FFPP complex and apoenzyme structures, suggesting that these residues provide interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L and F183A variants were observed to be severely restricted in their ability to catalyze C1-C11 cyclization of the FPP substrate and instead produced predominantly acyclic terpene products dominated by farnesol, β-farnesene, and nerolidol.
为了深入了解催化初始反马氏规则环化反应的萜烯合酶的反应机制，我们解析了 (+)-石竹烯-1-醇合酶（CS）的 X 射线晶体结构，其与非活性法呢基二磷酸（FPP）类似物 2-氟法呢基二磷酸（2FFPP）结合于酶的活性部位，以及没有结合 2FFPP 的 CS 结构。CS-2FFPP 结构解析至 2.65 Å 分辨率，显示配体处于拉长的构象，无法进行初始环化反应以形成 C1-C11 键。有趣的是，apo CS 结构（2.2 Å）在活性部位也有电子密度，在这种情况下，卷曲的四乙二醇分子很好地适合该位置，推测该分子来自结晶介质。该密度也很好地适合法呢烯分子，这表明该结构可能模拟反应坐标上的一个中间产物。与 2FFPP 结构相比，四乙二醇的卷曲构象伴随着一些活性部位残基的剧烈旋转。最值得注意的是，W56 和 F183 在 2FFPP 复合物和 apoenzyme 结构之间发生 90°旋转，这表明这些残基提供了相互作用，有助于卷曲四乙二醇分子在活性部位，并且可能在环化反应的正常过程中也卷曲 FPP 底物的衍生物。为了支持这一假设，CS W56L 和 F183A 变体观察到其催化 FPP 底物 C1-C11 环化的能力受到严重限制，反而主要产生以法呢醇、β-法呢烯和橙花叔醇为主的无环萜烯产物。
