Structural determination and characterisation of the CYP105Q4 cytochrome P450 enzyme from Mycobacterium marinum.

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

The binding of nitrogen-donor ligands to the ferric and ferrous forms of cytochrome P450 enzymes.

The cytochrome P450 superfamily of heme-thiolate monooxygenase enzymes can catalyse various oxidation reactions. The addition of a substrate or an inhibitor ligand induces changes in the absorption spectrum of these enzymes and UV-visible (UV-vis) absorbance spectroscopy is the most common and readily available technique used to interrogate their heme and active site environment. Nitrogen-containing ligands can inhibit the catalytic cycle of heme enzymes by interacting with the heme. Here we evaluate the binding of imidazole and pyridine-based ligands to the ferric and ferrous forms of a selection of bacterial cytochrome P450 enzymes using UV-visible absorbance spectroscopy. The majority of these ligands interact with the heme as one would expect for type II nitrogen directly coordinated to a ferric heme-thiolate species. However, the spectroscopic changes observed in the ligand-bound ferrous forms indicated differences in the heme environment across these P450 enzyme/ligand combinations. Multiple species were observed in the UV-vis spectra of the ferrous ligand-bound P450s. None of the enzymes gave rise to the isolation of a single species with a Soret band at ∼442-447 nm, indicative of a 6-coordinate ferrous thiolate species with a nitrogen-donor ligand. A ferrous species with Soret band at ∼427 nm coupled with an α-band of increased intensity was observed with the imidazole ligands. With some enzyme-ligand combinations reduction resulted in breaking of the iron­nitrogen bond yielding a 5-coordinate high-spin ferrous species. In other instances, the ferrous form was readily oxidised back to the ferric form on addition of the ligand.

A comparison of the bacterial CYP51 cytochrome P450 enzymes from Mycobacterium marinum and Mycobacterium tuberculosis.

Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.