Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity
Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity
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DOI：
10.1002/cbic.201900389
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发表时间：
2019-11-20
期刊：
影响因子：
3.2
通讯作者：
Guo, Zheng
中科院分区：
文献类型：
--
作者：
Eser, Bekir Engin;Poborsky, Michal;Guo, Zheng
脂肪酸水合酶(FAHS)对脂肪酸(FA)的酶促转化为生产高价值羟基脂肪酸(HFA)提供了一条绿色高效的途径。然而，到目前为止，在链长和羟基位置方面，HFA之间的多样性有限。本研究比较了嗜酸乳杆菌的两个高度相似的FHs：FA-HY2底物范围窄，区域选择性严格，而FA-HY1利用较长的链底物和水合物的不同双键位置。结果表明，三个活性中心残基对底物的特异性和水合反应的区域选择性具有显著的指导作用。如果将FA-HY2上的这些残基突变为FA-HY1中的相应残基，则底物范围显著扩大，双键向FA的omega-端的水合作用明显增强。FA-HY2的三个残基突变体(TM-FA-HY2)显示出令人印象深刻的对亚油酸的区域选择性逆转，使HFA区域异构体(10-OH/13-OH)的比例从99：1变为12：88。花生四烯酸和C-18多不饱和脂肪酸底物的区域选择性也有显著变化。此外，在制备规模上，TM-FA-HY2将二十碳五烯酸转化为12-羟基产物，转化率较高。此外，还证明了微藻是生产HFA的各种脂肪酸的来源。这项研究为量身定做的FAH设计铺平了道路，使其能够生产从聚合物工业到医疗领域的各种应用。
Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the omega-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C-18 polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.