Structural and Biochemical Studies of Substrate Selectivity in Ascaris suum Thiolases
Structural and Biochemical Studies of Substrate Selectivity in Ascaris suum Thiolases
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DOI：
10.1021/acs.biochem.7b01123
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发表时间：
2018-06-05
期刊：
影响因子：
2.9
通讯作者：
Chang, Michelle C. Y.
中科院分区：
文献类型：
--
作者：
Blaisse, Michael R.;Fu, Beverly;Chang, Michelle C. Y.
硫解酶是一类在生物技术和代谢工程中具有重要应用的碳-碳键形成酶，因为它们提供了用于缩合两个酰基辅酶A（CoA）底物的一般方法。因此，对它们的底物选择性有更深入的了解将扩大我们设计酶或微生物生产广泛的小分子靶点的能力。在这里，我们报告的晶体结构和生化特性的Acat 2和Acat 5，两个生物合成的硫解酶从猪蛔虫不同的选择性支化相比，线性化合物。结合丙酰辅酶A的Acat 2-C91 S突变体的结构表明，底物的末端甲基（代表α-分支点）指向保守的Phe 288和Met 158残基。在Acat 5中，Phe环旋转以适应与相邻Thr侧链的羟基-pi相互作用，减少结合口袋中的空间，并可能解释其与Acat 2相比对线性底物的强烈偏好。不同的Acat硫解酶结构的比较表明，Met 158是灵活的，采用交替的构象与侧链旋转朝向或远离覆盖环在后面的活性位点。Acat 5中的覆盖环中的残基与来自Acat 2的相应残基的突变允许高度增加的分支底物的容纳，而匡威的突变不显著影响Acat 2底物选择性。我们的研究结果表明，第二壳残基的硫解酶底物的选择性的重要贡献，并提供深入了解工程这类酶。
Thiolases are a class of carbon-carbon bond forming enzymes with important applications in biotechnology and metabolic engineering as they provide a general method for the condensation of two acyl coenzyme A (CoA) substrates. As such, developing a greater understanding of their substrate selectivity would expand our ability to engineer the enzymatic or microbial production of a broad range of small-molecule targets. Here, we report the crystal structures and biochemical characterization of Acat2 and Acat5, two biosynthetic thiolases from Ascaris suum with varying selectivity toward branched compared to linear compounds. The structure of the Acat2-C91S mutant bound to propionyl-CoA shows that the terminal methyl group of the substrate, representing the alpha-branch point, is directed toward the conserved Phe 288 and Met 158 residues. In Acat5, the Phe ring is rotated to accommodate a hydroxyl-pi interaction with an adjacent Thr side chain, decreasing space in the binding pocket and possibly accounting for its strong preference for linear substrates compared to Acat2. Comparison of the different Acat thiolase structures shows that Met 158 is flexible, adopting alternate conformations with the side chain rotated toward or away from a covering loop at the back of the active site. Mutagenesis of residues in the covering loop in Acat5 with the corresponding residues from Acat2 allows for highly increased accommodation of branched substrates, whereas the converse mutations do not significantly affect Acat2 substrate selectivity. Our results suggest an important contribution of second-shell residues to thiolase substrate selectivity and offer insights into engineering this enzyme class.