醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响

Effect of position 127 on the activity and enantioselectivity of alcohol dehydrogenase KpADH

醇脱氢酶可用于合成手性化合物,在医药、材料等领域应用广泛。来源于多孢克鲁维酵母(Kluyveromyces polysporus)的醇脱氢酶KpADH同时具有还原(4-氯苯基)-(吡啶-2-基)-甲酮(CPMK)和氧化异丙醇、1,4-丁二醇的活性。通过分子对接和结构分析,发现Y127是紧靠底物的关键位点。本文通过对127位点定点饱和突变来提高催化活性和对映选择性,突变体Y127V和Y127I还原CPMK的比活力达到95.0U/mg和84.0U/mg,分别是野生型的6.5倍和5.8倍,突变体Y127L催化CPMK生成(R)-(4-氯苯基)-(吡啶-2-基)-甲醇[(R)-CPMA]的e.e.值由82%提高到99.2%。同时,127位点的突变提高了对醇类底物的氧化活性,突变体Y127I对异丙醇的比活力是野生型的1.46倍,而Y127C更青睐于对1,4-丁二醇的催化氧化,其比活力是野生型的3.00倍。分子间作用力分析表明,Y127L与底物CPMK间增加的氢键和π-π作用力稳定了底物构象,进一步提高了还原CPMK的对映选择性。本文为醇脱氢酶KpADH的分子改造和机制解析提供了指导,提高了该酶的工业应用潜力。

Alcohol dehydrogenases can be used to synthesize chiral compounds, which are widely applied in pharmaceuticals, materials and other fields. Alcohol dehydrogenase from Kluyveromyces polysporus(KpADH) exhibited both reducing activity toward(4-chlorophenyl)-(pyridin-2-yl)-methanone(CPMK)and oxidizing activity toward isopropanol and 1,4-butanediol. Based on molecular docking and structural analysis, Y127 was identified as the key site close to the substrate. To improve the catalytic performance and enantioselectivity, site-saturation mutagenesis was applied at Y127. Specific activity toward CPMK of variants Y127 V and Y127 I increased to 95.0 U/mg and 84.0 U/mg, which were 6.5-and 5.8-fold of wild type KpADH(WTKpADH), respectively. The enantioselectivity toward CPMK of variant Y127 L increased from 82% to 99.2% e. e.(R). Mutations at 127 also enhanced the oxidizing activity toward alcohol substrates. Specific activity toward isopropanol of Y127 I was 1.46-fold ofWTKpADH. Variant Y127 C displayed higher activity in the oxidation of 1,4-butanediol, whose specific activity was 3.00-fold of WTKpADH. Intermolecular forces analysis indicated that an increased hydrogen bond and extra π-πinteractions stabilized the conformation, which resulted in enhanced enantioselectivity. This study provides guidance for molecular engineering and mechanism analysis of alcohol dehydrogenase KpADH,and extends its application potential in industry.