Structural and Functional Characterisation of a Metagenome-Derived CYP108D18 Reveals an Unusual Dioxygen-Bound State and Selective Hydrocarbon Hydroxylation.

Authors

Simran Kundral, Peter D Giang, Alicia M Kirk, Julia B Buczynski, Kasun S A Arachchige, Sunil K Khare, Paul V Bernhardt, Paul N Evans, Luke W Guddat, Stephen G Bell, James J De Voss

Published in

Microbial biotechnology. Volume 19. Issue 10. Pages e70451.

Abstract

Cytochrome P450 enzymes form a large superfamily of monooxygenases capable of oxidising diverse substrates. Among them, members of the CYP108 family are recognised for their role in monoterpenoid oxidation. In this study, we report the identification and characterisation of a new CYP108 family member, CYP108D18, from a metagenome-assembled genome derived from microbial communities in a geothermal hot spring located in northern Queensland, Australia. Taxonomic binning associated CYP108D18 with a Novosphingobium species, a genus well-known for its bioremediation potential. CYP108D18 was heterologously expressed in Escherichia coli, yielding 640 nmol L^-1 of culture, and purified to homogeneity. The enzyme exhibited moderate thermostability, with a ¹⁵T₅₀ value of 47°C ± 1°C. Spectroscopic substrate binding assays revealed that while CYP108D18 bound monoterpenoids similarly to other CYP108 enzymes, it showed higher affinity and pronounced larger heme spin-state shifts with aromatic hydrocarbons such as phenylcyclohexane and phenanthrene. Subsequent in silico docking and spectral-binding studies also suggested that CYP108D18 may accommodate larger plant-derived sesquiterpenes, indicating a potential role in their metabolism. Reconstitution of CYP108D18 with spinach ferredoxin and ferredoxin reductase resulted in the oxidation of phenylcyclohexane to trans-4-phenylcyclohexanol. Molecular dynamics simulations and ONIOM (QM/MM) calculations supported selective hydrogen abstraction at the cyclohexane C4 position, rationalising trans-4-phenylcyclohexanol as the predominant product. The high-resolution crystal structure of substrate-free CYP108D18 revealed a bound dioxygen ligand positioned above the heme iron, an unusual feature among P450 structures. Both substrate-free and phenylcyclohexane-bound structures revealed a persistent open conformation, suggesting the absence of a canonical open-to-closed transition upon ligand binding. Spectroelectrochemical analysis showed a heme redox potential of -451 mV versus NHE for the substrate-free CYP108D18, shifting subtly to -409 mV upon phenylcyclohexane binding, which contributes to reduced activity with non-native [2Fe-2S] ferredoxins.

PMID:
42817572
Bibliographic data and abstract were imported from PubMed on 01 Oct 2026.

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