The 5′-hydroxymethyl metabolite of the penicillin based antibiotic flucloxacillin (FLX) is considered to be involved in bile duct damage occurring in a small number of patients. Because 5′-hydroxymethyl FLX is difficult to obtain by organic synthesis, biosynthesis using highly active and regioselective biocatalysts would be an alternative approach. By screening an in-house library of Cytochrome P450 (CYP) BM3 mutants, mutant M11 L437E was identified as a regioselective enzyme with relatively high activity in production of 5′-hydroxymethyl FLX as was confirmed by mass spectrometry and NMR. In contrast, incubation of M11 L437E and other mutants with oxacillin (OX, which differs from FLX by a lack of aromatic halogens) resulted in formation of two metabolites. In addition to 5′-hydroxymethyl OX we identified a product resulting from aromatic hydroxylation. In silico studies of both FLX and OX with three CYP BM3 mutants revealed substrate binding poses allowing for 5′-methyl hydroxylation, as well as binding poses with the aromatic moiety in the vicinity of the heme iron for which the corresponding product of aromatic hydroxylation was not observed for FLX. Supported by the (differences in) experimentally determined ratios of product formation for OX hydroxylation by M11 and its L437A variant and M11 L437E, Molecular Dynamics simulations suggest that the preference of mutant M11 L437E to bind FLX in its catalytically active pose over the other binding orientation contributes to its biocatalytic activity, highlighting the benefit of studying effects of active-site mutations on possible alternative enzyme-substrate binding poses in protein engineering.

基于青霉素的抗生素氟氯西林（FLX）的5'-羟甲基代谢产物被认为与少数患者发生的胆管损伤有关。由于难以通过有机合成获得5'-羟甲基FLX，因此使用高活性和区域选择性生物催化剂进行生物合成将是一种替代方法。通过筛选细胞色素P450（CYP）BM3突变体的内部文库，突变体M11 L437E如通过质谱法和NMR所证实的，“α-β”被鉴定为在5'-羟甲基FLX的产生中具有相对较高活性的区域选择性酶。相反，将M11 L437E和其他突变体与奥沙西林（OX，由于缺乏芳香族卤素而不同于FLX）孵育，导致形成两种代谢物。除5'-羟甲基OX外，我们还鉴定出由芳族羟基化产生的产物。在计算机上对具有三个CYP BM3突变体的FLX和OX的研究表明，底物结合姿势允许5'-甲基羟基化，以及与血红素铁附近的芳香族部分的结合姿势（未观察到相应的芳香族羟基化产物）对于FLX。由实验确定的M11及其L437A变体和M11 L437E的OX羟基化形成的比率（存在差异）支持，分子动力学模拟表明，突变体M11 L437E在其催化活性位置结合FLX的偏好高于其他结合方向有助于其生物催化活性，突出了研究活性位点突变对蛋白质工程中可能的替代酶-底物结合姿势的影响的益处。