Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering.
Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering.
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DOI：
10.1038/s41598-018-28177-4
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发表时间：
2018-07-02
影响因子：
4.6
通讯作者：
Quax WJ
中科院分区：
文献类型：
--
作者：
Abdallah II;van Merkerk R;Klumpenaar E;Quax WJ
amorpha4,11-二烯合成酶（ADS）环化底物法尼基焦磷酸，生成amorpha4,11-二烯作为主要产物。这被认为是抗疟青蒿素生物合成的第一个坚定和限速步骤。在这里，我们利用已报道的ADS三维模型来执行易变性景观引导酶工程。建立了一个包含16个活性位点残基的258个突变体文库，并对其催化活性和产物谱进行了筛选。这样就可以确定其中一些残基在机制中的作用。R262限制释放的焦磷酸基团和镁离子。芳香残基（W271， Y519和F525）稳定中间碳正离子，而T296， G400， G439和L515则有助于1,6和1,10环的闭合。最后，建议W271与T399一起作为活性位点碱基，以确保区域选择性去质子化。可变性景观也有助于确定具有改进催化活性的变体。H448A的催化效率提高了4倍，双突变T399S/H448A的催化效率提高了5倍。这一变异可用于促进青蒿素的生物合成。我们的发现为提高青蒿素的工业生产迈出了第一步，并为进一步的工程和理解ADS开辟了可能性。
Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved kcat by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.
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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
Amorpha-4,11-二烯合酶的 T296V 突变体在烯丙基二磷酸异构化方面存在缺陷，但保留了将中间体 (3R)-橙花烷基二磷酸环化为 Amorpha-4,11-二烯的能力。
影响因子：
2.9
作者：
Li, Zhenqiu;Gao, Ruiping;Hao, Qinggang;Zhao, Huifang;Cheng, Longbin;He, Fang;Liu, Li;Liu, Xiuhua;Chou, Wayne K. W.;Zhu, Huajie;Cane, David E.
通讯作者：
Cane, David E.
影响因子：
4
作者：
Noel, Joseph P.;Dellas, Nikki;Faraldos, Juan A.;Zhao, Marylin;Hess, B. Andes, Jr.;Smentek, Lidia;Coates, Robert M.;O'Maille, Paul E.
通讯作者：
O'Maille, Paul E.
影响因子：
8
作者：
Diaz, Juan E.;Lin, Chun-Shi;Weiss, Gregory A.
通讯作者：
Weiss, Gregory A.
影响因子：
11.9
作者：
Gao Y;Honzatko RB;Peters RJ
通讯作者：
Peters RJ