Department of Chemistry, University of Adelaide, SA 5005, Australia.
School of Biological Sciences, University of Adelaide, SA 5005, Australia.
Biochim Biophys Acta Gen Subj. 2019 May;1863(5):925-934. doi: 10.1016/j.bbagen.2019.02.016. Epub 2019 Feb 28.
Actinobacteria, including the Mycobacteria, have a large component of cytochrome P450 family monooxygenases. This includes Mycobacterium tuberculosis, M. ulcerans and M. marinum, and M. vanbaalenii. These enzymes can abstract CH bonds and have important roles in natural product biosynthesis.
Two members of the bacterial CYP150 family, CYP150A5 and CYP150A6 from M. marinum, were produced, purified and characterised. The potential substrate ranges of both enzymes were analysed and the monooxygenase activity of CYP150A5 was reconstituted using a physiological electron transfer partner system. CYP150A6 was structurally characterised by X-ray crystallography.
CYP150A5 was shown to bind various norisoprenoids and terpenoids. It could regioselectively hydroxylate β-ionol. The X-ray crystal structure of substrate-free CYP150A6 was solved to 1.5 Å. This displayed an open conformation with short F and G helices, an unresolved F-G loop region and exposed active site pocket. The active site residues could be identified and important variations were found among the CYP150A enzymes. Haem-binding azole inhibitors were identified for both enzymes.
The structure of CYP150A6 will facilitate the identification of physiological substrates and the design of better inhibitors for members of this P450 family. Based on the observed differences in substrate binding preference and sequence variations among the active site residues, their roles are predicted to be different.
Multiple CYP150 family members were found in many bacteria and are prevalent in the Mycobacteria including several human pathogens. Inhibition and structural data are reported here for these enzymes for the first time.
放线菌包括分枝杆菌，其细胞色素 P450 家族单加氧酶有很大一部分。这包括结核分枝杆菌、溃疡分枝杆菌和海分枝杆菌，以及 M. vanbaalenii。这些酶可以提取 CH 键，并在天然产物生物合成中具有重要作用。
从海分枝杆菌中产生、纯化并表征了细菌 CYP150 家族的两个成员，CYP150A5 和 CYP150A6。分析了这两种酶的潜在底物范围，并使用生理电子转移伙伴系统重建了 CYP150A5 的单加氧酶活性。通过 X 射线晶体学对 CYP150A6 进行了结构表征。
CYP150A5 被证明可以结合各种异戊二烯和萜烯。它可以选择性地羟化 β-依兰醇。未结合底物的 CYP150A6 的 X 射线晶体结构解决到 1.5Å。这显示了一个开放的构象，短 F 和 G 螺旋，未解决的 F-G 环区和暴露的活性位点口袋。可以识别活性位点残基，并在 CYP150A 酶中发现了重要的变异。鉴定了两种酶的血红素结合唑抑制剂。
CYP150A6 的结构将有助于确定生理底物，并为该 P450 家族的成员设计更好的抑制剂。基于观察到的在底物结合偏好和活性位点残基中的序列变异方面的差异，预测它们的作用是不同的。
许多细菌中都发现了多种 CYP150 家族成员，包括几种人类病原体中的分枝杆菌。本文首次报道了这些酶的抑制和结构数据。
