Investigating the applicability of the CYP102A1-decoy-molecule system to other members of the CYP102A subfamily

In the endeavour to develop green catalysts for the transformation of organomolecules, cytochrome P450 enzymes (CYPs) have emerged as promising candidates. Most notably, the self-sufficient fatty acid monooxygenase CYP102A1 from Priestia megaterium (formerly Bacillus megaterium, Fig. 1a) has emerged at the forefront of this endeavour, owing to its unrivalled high catalytic activity for its native substrate, long-chain fatty acids. The general catalytic cycle for P450 enzymes [1] is depicted in Fig. 1b. In the first step, the substrate (RH) binds to the active site, removing a water molecule that is ligated to a molecule of haem located in the active-site cavity of CYP102A1 (I → II). This causes a positive shift in the redox potential of the haem iron, permitting the uptake of electrons from NADPH via the reductase domain (II → III). Subsequent binding (III → IV) and reduction (IV → VII) of dioxygen culminate in the formation of the active species, Compound I (Cpd I, VII), which possesses the power to oxidise a variety of Csingle bondH bonds (VII → IX). The trade-off for the high catalytic activity of CYP102A1 and related P450 enzymes is a narrow substrate specificity, thus, CYP102A1 generally does not hydroxylate non-native substrates, such as small alkanes and aromatics, as they are incapable of activating the enzyme (Fig. 1c). To overcome this limitation, many researchers have exploited powerful techniques such as site-directed mutagenesis and directed evolution to change the substrate specificity of CYP102A1 [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]]. As an alternative approach, our research group has developed a system that does not require the use of mutagenesis, having the great benefit of working with the wild-type enzyme [13].

This approach makes use of substrate mimics (decoy molecules) that resemble the native substrate but are not converted themselves. Employing such decoy molecules, CYP102A1 can be “tricked” into becoming active and hydroxylating non-native substrates, such as propane and benzene. The decoy molecule achieves this much like the native substrate by first dislodging haem-bound water, which stimulates the generation of Cpd I. Furthermore, the decoy molecule helps mould the active site for non-native substrate binding and oxidation (Fig. 1d). Recent reviews describing the minutiae of this system can be accessed here [[14], [15], [16], [17], [18]]. Since its inception over a decade ago, the CYP102A1-decoy-molecule system has been developed extensively, whilst other members of the extensive CYP102 family have gone largely ignored. To show the broader applicability of this method for other CYP102 family enzymes, we have selected two closely related P450s from the CYP102A subfamily, namely CYP102A5 from Bacillus cereus and CYP102A7 from Bacillus licheniformis.

Herein, we demonstrate that decoy molecules can indeed be used with CYP102-family enzymes other than CYP102A1 and observe striking differences in decoy molecule selectivity between the three CYP102A haemoenzymes. Furthermore, we show that CYP102A5 and A7 express aberrant binding behaviour differing significantly from the CYP102A1 reference system. The X-ray crystal structure of the CYP102A5 haem domain was also successfully solved, offering insights into a potential substrate-binding site of CYP102A5.

留言 (0)

沒有登入
gif