An Aromatic Cluster in the Active Site of epi-Isozizaene Synthase Is an Electrostatic Toggle for Divergent Terpene Cyclization Pathways.
An Aromatic Cluster in the Active Site of epi-Isozizaene Synthase Is an Electrostatic Toggle for Divergent Terpene Cyclization Pathways.
复制标题
表异齐烯合成酶活性中心的芳香簇是萜烯环化途径的静电触发器。
DOI：
10.1021/acs.biochem.0c00876
复制
发表时间：
2020-12-22
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：
Ronnebaum TA;Gardner SM;Christianson DW
倍半萜环化酶epi-isozizaene合酶（EIZS）催化法呢基二磷酸的环化以形成抗生素albaflavenone的三环前体。疏水活性位点主要由芳族残基定义，其通过多个碳阳离子中间体指导多步反应序列。之前用极性残基取代关键的芳香残基F96，将EIZS转化为高保真倍半卡宾合酶：F96 S、F96 M和F96 Q变体分别产生78%、91%和97%的倍半卡宾A。在这里，我们报告高分辨率的X射线晶体结构的两个重编程环化酶。F96 M E112-Mg 2 +3-利塞膦酸盐和F96 M E112-Mg 2 +3-无机焦磷酸盐-苄基三乙基铵阳离子络合物的结构揭示了F96芳族簇中的结构变化，其改变了催化中红没药基碳阳离子中间体导致的环化途径。F96 S EIBR-Mg 2 +3-奈立膦酸盐复合物的结构揭示了部分占据的抑制剂和在开放状态和闭合状态之间的转变中捕获的酶活性位点。最后，野生型EIZS与双膦酸盐抑制剂奈立膦酸盐、帕米膦酸盐和利塞膦酸盐复合的三种结构为理解野生型和变体酶之间的结合差异提供了基础。这些结构提供了关于活性位点灵活性的新见解，特别是关于微妙的膨胀和收缩以容纳不同大小的配体以及结合水分子的潜力。此外，这些结构突出了F96芳族簇中构象变化的重要性，这些构象变化可能影响催化中阳离子与碳阳离子中间体的阳离子-π相互作用。
The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic precursor of the antibiotic albaflavenone. The hydrophobic active site is largely defined by aromatic residues that direct a multistep reaction sequence through multiple carbocation intermediates. The previous substitution of polar residues for a key aromatic residue, F96, converts EIZS into a high-fidelity sesquisabinene synthase: the F96S, F96M, and F96Q variants respectively generate 78%, 91%, and 97% sesquisabinene A. Here, we report high-resolution X-ray crystal structures of two of these reprogrammed cyclases. The structures of the F96M EIZS–Mg2+3–risedronate and F96M EIZS–Mg2+3–inorganic pyrophosphate-benzyltriethylammonium cation complexes reveal structural changes in the F96 aromatic cluster that redirect the cyclization pathway leading from the bisabolyl carbocation intermediate in catalysis. The structure of the F96S EIZS–Mg2+3–neridronate complex reveals a partially occupied inhibitor and an enzyme active site caught in transition between open and closed states. Finally, three structures of wild-type EIZS complexed with the bisphosphonate inhibitors neridronate, pamidronate, and risedronate provide a foundation for understanding binding differences between wild-type and variant enzymes. These structures provide new insight regarding active site flexibility, particularly with regard to the potential for subtle expansion and contraction to accommodate ligands of varying sizes as well as bound water molecules. Additionally, these structures highlight the importance of conformational changes in the F96 aromatic cluster that could influence cation-π interactions with carbocation intermediates in catalysis.
登录
查看更多内容
DOI：
10.1107/s0907444909029436
发表时间：
2009-10-01
影响因子：
2.2
作者：
Moriarty, Nigel W.;Grosse-Kunstleve, Ralf W.;Adams, Paul D.
通讯作者：
Adams, Paul D.
影响因子：
2.9
作者：
Li R;Chou WK;Himmelberger JA;Litwin KM;Harris GG;Cane DE;Christianson DW
通讯作者：
Christianson DW
影响因子：
2.9
作者：
Blank PN;Barrow GH;Chou WKW;Duan L;Cane DE;Christianson DW
通讯作者：
Christianson DW
影响因子：
5.6
作者：
Andre CM;Hausman JF;Guerriero G
通讯作者：
Guerriero G
影响因子：
3.8
作者：
Bleeker, Petra M.;Diergaarde, Paul J.;Schuurink, Robert C.
通讯作者：
Schuurink, Robert C.