Structural studies of an A2-type modular polyketide synthase ketoreductase reveal features controlling α-substituent stereochemistry.
Structural studies of an A2-type modular polyketide synthase ketoreductase reveal features controlling α-substituent stereochemistry.
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DOI：
10.1021/cb400161g
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发表时间：
2013-09-20
影响因子：
4
通讯作者：
Keatinge-Clay, Adrian T.
中科院分区：
文献类型：
--
作者：
Zheng, Jianting;Piasecki, Shawn K.;Keatinge-Clay, Adrian T.
当还原复杂聚酮化合物生物合成中的中间体时，模块化聚酮化合物合酶酮还原酶通常设置两个立体中心。在这里，我们报告了来自两性霉素聚酮合酶第十一个模块的A2型酮还原酶的2.60 Å分辨率结构，该酶设定了l-α-甲基和l-β-羟基立体化学的组合，并代表了有待结构阐明的最终具有催化能力的酮还原酶类型。通过结构引导诱变，产生了 A1 型酮还原酶的双突变体，其在二酮化合物底物类似物上充当 A2 型酮还原酶，将 α-烷基取代基设置为 l 方向，而不是未突变的酮还原酶设置的 d 方向。当在工程化的三酮化合物内酯合酶的背景下检查双突变体的活性时，没有产生预期的三酮化合物内酯，即使含有酮还原酶的模块仍然如预期地还原二酮化合物底物类似物。这些发现表明，重新设计的酮还原酶可能在工程化的聚酮合酶装配线中在催化方面被击败。
Modular polyketide synthase ketoreductases often set two stereocenters when reducing intermediates in the biosynthesis of a complex polyketide. Here we report the 2.60 Å-resolution structure of an A2-type ketoreductase from the eleventh module of the amphotericin polyketide synthase that sets a combination of l-α-methyl and l-β-hydroxyl stereochemistries and represents the final catalytically-competent ketoreductase type to be structurally elucidated. Through structure-guided mutagenesis a double mutant of an A1-type ketoreductase was generated that functions as an A2-type ketoreductase on a diketide substrate analog, setting an α-alkyl substituent in an l- orientation rather than in the d-orientation set by the unmutated ketoreductase. When the activity of the double-mutant was examined in the context of an engineered triketide lactone synthase, the anticipated triketide lactone was not produced even though the ketoreductase-containing module still reduced the diketide substrate analog as expected. These findings suggest that re-engineered ketoreductases may be catalytically outcompeted within engineered polyketide synthase assembly lines.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.2
作者：
Caffrey, P
通讯作者：
Caffrey, P
影响因子：
--
作者：
Baerga-Ortiz, A;Popovic, B;Leadlay, PF
通讯作者：
Leadlay, PF
影响因子：
4.1
作者：
Dutta, Debajyoti;Bhattacharyya, Sudipta;Das, Amit Kumar
通讯作者：
Das, Amit Kumar
影响因子：
2.7
作者：
Sharma, Krishna K.;Boddy, Christopher N.
通讯作者：
Boddy, Christopher N.