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首页> 外文期刊>Biochemistry >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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Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes

机译：ePI-异氮化酶合酶的活性位点袋中的极性残留物替代芳族残基导致新的循环倍半萜烯的产生

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摘要

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 eight 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 ndnpolar 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.
机译：SesquiterPene环酶EPI-异氮化酶合酶（eIZ）催化法呢基二磷酸的环化以形成抗生素阿尔法酮的三环烃前体。 EIZ的疏水性活性位点袋用作模板，因为它绑定和伴侣源柔性基板和碳粉座中间体通过多步骤反应序列所需的构象。我们以前表明，用其他疏水性残留物替代疏水性残留物恢复模板和扩展产品化学大学[Li，R.，Chou，WKW，Himmelberger，Ja，Litwin，Km，Harris，GG，Cane，De和Christianson，DW （2014）Biochemistry 53,1155-1168]。这里，我们表明疏水性残留物特异性，Y69，F95，F96和W203 - 具有极侧链的替代也产生膨胀产品化学大学的官能酶催化剂。据报道，14个新的eizs突变体产生产品阵列，其中已识别出八种新的SesquitePene产品。值得注意的是，一些突变体产生无环和环状的羟基化产品，表明疏水袋中的极性引入促进了能够淬火碳粉型中间体的水的结合。此外，F96的极性残留物的取代产生高保真性倍二异烯烯烯合成酶。所选突变体的晶体结构揭示了定义疏水口袋的三维轮廓的残基可以被替代，而不会在活性位点其他地方触发显着的结构变化。因此，当Terpenoid环偶活性位点通过诱变和扩大产品化学大学的目标重新梳形时，应考虑更多基团NdnPolar极性氨基酸取代。

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  • 来源
    《Biochemistry》 |2017年第43期|共14页
  • 作者

    Blank Patrick N.; Barrow Golda H.; Chou Wayne K. W.; Duan Lian; Cane David E.; Christianson David W.;

  • 作者单位

    Univ Penn Dept Chem Roy &

    Diana Vagelos Labs Philadelphia PA 19104 USA;

    Univ Penn Dept Chem Roy &

    Diana Vagelos Labs Philadelphia PA 19104 USA;

    Brown Univ Dept Chem Box H Providence RI 02912 USA;

    Brown Univ Dept Chem Box H Providence RI 02912 USA;

    Brown Univ Dept Chem Box H Providence RI 02912 USA;

    Univ Penn Dept Chem Roy &

    Diana Vagelos Labs Philadelphia PA 19104 USA;

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  • 正文语种 eng
  • 中图分类 生物化学;
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