The cytochrome P450 enzymes CYP101B1 and CYP101C1, which are from the bacterium Novosphingobium aromaticivorans DSM12444, can hydroxylate norisoprenoids with high activity and selectivity. With the goal of expanding and establishing their substrate range with a view to developing applications, the oxidation of a selection of cyclic alkanes, ketones and alcohols was investigated. Cycloalkanes were oxidised, but both enzymes displayed moderate binding affinity and low levels of productive activity. We improved the binding and activity of these substrates with CYP101B1 by making the active site more hydrophobic by switching a histidine residue to a phenylalanine (H85F). The presence of a ketone moiety in the cycloalkane skeleton significantly improved the oxidation activity with both enzymes. CYP101C1 preferably catalysed the oxidation of cycloalkanones at the C-2 position whereas CYP101B1 oxidised these substrates with higher productivity and at positions remote from the carbonyl group. This demonstrates that the binding orientation of the cyclic ketones in the active site of each enzyme must be different. Linear ketones were also oxidised by both enzymes but with lower activity and selectivity. Cyclic substrates with an ester directing group were more efficiently oxidised by CYP101B1 than CYP101C1. Both enzymes catalysed oxidation of these esters with high regioselectively on the ring system remote from the ester directing group. CYP101C1 selectively oxidised certain terpenoid ester substrates, such as α-terpinyl and citronellyl acetate more effectively than CYP101B1. Overall, we establish that the high selectivity and activity of these enzymes could provide new biocatalytic routes to important fine chemicals.

中文翻译：

---

相同家族的两种细胞色素P450酶对烃衍生物的互补和选择性氧化

细胞色素P450酶CYP101B1和CYP101C1，来自细菌新孢子虫DSM12444可高活性和选择性地羟基化类异戊二烯。为了扩展和建立其底物范围，以开发应用，研究了选择环烷烃，酮和醇的氧化。环烷被氧化，但两种酶均显示出中等的结合亲和力和较低的生产活性。我们通过将组氨酸残基切换为苯丙氨酸（H85F），使活性位点更具疏水性，从而改善了这些底物与CYP101B1的结合和活性。环烷烃骨架中酮部分的存在显着改善了两种酶的氧化活性。CYP101C1优选在C-2位置催化环烷酮的氧化，而CYP101B1在远离羰基的位置以较高的生产率氧化这些底物。这表明每种酶的活性位点中环酮的结合方向必须不同。线性酮也被两种酶氧化，但活性和选择性较低。CYP101B1比CYP101C1更有效地氧化具有酯导向基团的环状底物。两种酶都在远离酯导向基团的环系统上以高区域选择性催化这些酯的氧化。CYP101C1比CYP101B1更有效地选择性氧化某些萜类酯底物，例如α-萜品基和香茅酯。总体，