Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes.
Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes.
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DOI：
10.1021/acs.biochem.7b00895
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发表时间：
2017-10-31
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：
Blank PN;Barrow GH;Chou WKW;Duan L;Cane DE;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的三环烃前体。EIZS的疏水活性位点口袋作为模板，因为它通过多步反应序列所需的构象结合和陪伴柔性底物和碳阳离子中间体。我们先前证明，用其它疏水残基取代疏水残基重塑模板并扩大产物化学多样性[Li，R.，Chou，W. K. W.，Himmelberger，J. A.，Litwin，K. M.，哈里斯，G。G.，Cane，D. E、Christianson，D. W.（2014）Biochemistry 53，1155-1168]。在这里，我们表明，取代疏水残基-特别是，Y 69，F95，F96和W203 -与极性侧链也产生功能酶催化剂，扩大产品的化学多样性。报道了14种新的EIZS突变体，其产生的产物阵列中已鉴定出8种新的倍半萜产物。值得注意的是，一些突变体产生无环和环状羟基化产物，这表明在疏水口袋中引入极性促进了能够淬灭碳阳离子中间体的水的结合。此外，极性残基取代F96产生高保真倍半卡宾烯酶。所选突变体的晶体结构表明，定义疏水口袋的三维轮廓的残基可以被取代，而不会在活性位点的其他地方引发显着的结构变化。因此，更多的激进的非极性极性氨基酸取代时，应考虑萜类化合物环化酶活性位点通过诱变改造的目标，探索和扩大产品的化学多样性。
The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) Biochemistry 53, 1155–1168]. Here, we show that the substitution of hydrophobic residues – specifically, Y69, F95, F96, and W203 – with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which 8 new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar-polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity.
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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：
3.8
作者：
Bleeker, Petra M.;Diergaarde, Paul J.;Schuurink, Robert C.
通讯作者：
Schuurink, Robert C.
影响因子：
11.9
作者：
Gao Y;Honzatko RB;Peters RJ
通讯作者：
Peters RJ
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
6.8
作者：
Chen, Mengbin;Harris, Golda G.;Pemberton, Travis A.;Christianson, David W.
通讯作者：
Christianson, David W.