Carvalho Alexandra T P, Dourado Daniel F A R, Skvortsov Timofey, de Abreu Miguel, Ferguson Lyndsey J, Quinn Derek J, Moody Thomas S, Huang Meilan
School of Chemistry and Chemical Engineering, Queen's University, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK.
Phys Chem Chem Phys. 2018 Jan 24;20(4):2558-2570. doi: 10.1039/c7cp07172h.
Phenylacetone monooxygenase is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenases family, and therefore it is an ideal candidate for the synthesis of industrially relevant ester or lactone compounds. However, its limited substrate scope has largely limited its industrial applications. Linear substrates are interesting from an industrial point of view, it is thus necessary to identify the essential spatial requirement for achieving high conversions for non-native linear substrates. Here using molecular dynamics simulations, we compared the conversion of a non-native linear substrate 2-octanone and the native substrate phenylacetone, catalyzed by the WT enzyme and a quadruple variant P253F/G254A/R258M/L443F that exhibits significantly improved activity towards 2-octanone. We uncovered that a remarkable movement of L289 is crucial for a reshaping of the active site of the quadruple variant so as to prevent the aliphatic substrate from moving away from the C4a-peroxyflavin, thus enabling it to keep a catalytically relevant pose during the oxygenation process. By performing steady-state kinetic analysis of two single-mutation variants at position 258, we further validated that the L289 reposition is attributed to the combined effect of quadruple mutations. In order to further explore the substrate scope of PAMO we also studied the binding of cyclopentanone and 2-phenylcyclohexanone, which are the typical substrates of CPMO in group I and CHMO in group III, respectively. Our study provides fundamental atomic-level insights in rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.
苯丙酮单加氧酶是拜耳-维利格单加氧酶家族中最稳定且耐热的成员，因此它是合成工业相关酯或内酯化合物的理想候选者。然而，其有限的底物范围在很大程度上限制了其工业应用。从工业角度来看，线性底物很有吸引力，因此有必要确定实现非天然线性底物高转化率所需的基本空间要求。在这里，我们使用分子动力学模拟，比较了野生型酶和对2-辛酮表现出显著提高活性的四重变体P253F/G254A/R258M/L443F催化非天然线性底物2-辛酮和天然底物苯丙酮的转化情况。我们发现L289的显著移动对于四重变体活性位点的重塑至关重要，从而防止脂肪族底物从C4a-过氧黄素移开，使其在氧化过程中能够保持催化相关的构象。通过对258位的两个单突变变体进行稳态动力学分析，我们进一步验证了L289的重新定位归因于四重突变的综合作用。为了进一步探索PAMO的底物范围，我们还研究了环戊酮和2-苯基环己酮的结合情况，它们分别是I组CPMO和III组CHMO的典型底物。我们的研究为PAMO的合理工程改造提供了基本的原子水平见解，以便在工业生物催化中广泛应用，特别是在将长链脂肪族油生物转化为潜在生物柴油方面。
