The cytochromes P450 (P450s) are ubiquitous haem-containing metalloenzymes that catalyse highly selective oxidation reactions across a broad range of organic substrates throughout nature. In bacteria, P450s commonly initiate the hydroxylation of organic compounds, enabling a cascade of downstream catabolic pathways for the degradation of natural products. While these enzymes have a common overall fold, their vast substrate scope is controlled by significant and bespoke changes in the architecture of the active site cavity which bind and position the substrate. These interactions are best revealed using X-ray crystallography. We have investigated the structure–function relationships of newly characterised bacterial P450 enzymes involved in diterpenoid, triterpenoid and steroid oxidation using high-resolution X-ray crystal structures in complex with these substrates. These compounds and their metabolites have useful biological activity but are also emerging contaminants and their oxidation could facilitate their removal from wastewater in the future.
Crystal structures of CYP226 enzymes from Zestomonas thermotolerans (CYP226A31) were determined in complex with all the major abietane-type ‘resin acid’ diterpenoids present in gum rosin, including abietic acid, dehydroabietic acid, pimaric acid, isopimaric acid and neoabietic acid at good resolution (1.65 – 1.98 Å). The Z. thermotolerans bacterium utilise these environmentally toxic substrates for growth and the enzyme may potentially have use in wastewater bioremediation. These structures were good enough to enable the different diterpenoid scaffolds to be distinguished. Conserved charged amino acids forming polar contacts with the substrate carboxylate moiety are especially important for rigidly holding resin acids in place in the active site. Comparison with substrate-free structures of CYP226A31 revealed conformational changes of active-site residues associated with substrate recognition and binding suitable for enantioselective catalysis. Structural comparison with equally good resolution (1.70 Å) homologous CYP226 enzymes from Caldimonas thermotolerans identified conserved structural motifs that underpin their comparable substrate and product selectivity and catalytic efficiency. The structures enable analysis of the solvent content of the enzymes to provide a structural rationale for the unusual UV-vis and peroxygenase activity.
To further investigate substrate recognition across more distantly related P450 families, a high-resolution (1.49 Å) x-ray crystal structure of a CYP106 enzyme from Thermoactinomyces vulgaris was solved in complex with a non-native substrate the oleane-type pentacyclic triterpenoid 17β-glycyrrhetinic acid. Triterpenes have potential for therapeutic applications however selective high-yielding C-H bond activation remains largely unexplored. The bulky substrate is accommodated into the large active site and loosely held by few polar interactions and remains structurally flexible, providing insight into the versatile substrate range and low substrate and product selectivity observed by CYP106 enzymes.
Finally, the structure of CYP1750B2 from Caenibius tardaugens was determined in complex with the steroid hormone 17β-oestradiol (2.88 Å). Important residues responsible for substrate recognition and positioning tailored for aromatic hydroxylation to produce only the 4-hydroxy oestradiol product were observed.
These structures provide insights into the molecular basis of substrate specificity and ligand recognition in bacterial P450 enzymes. The identification of conserved structural motifs and substrate-binding determinants enhance our understanding of P450 catalysis and provides a structural framework for future enzyme engineering and biocatalytic applications.