The Structures of the Steroid Binding CYP142 Cytochrome P450 Enzymes from Mycobacterium ulcerans and Mycobacterium marinum

Abstract

The steroid binding CYP142 cytochrome P450 enzymes of Mycobacterium species are involved in the metabolism of cholesterol and its derivatives. The equivalent enzyme from Mycobacterium ulcerans was studied to compare the degree of functional conservation between members of this CYP family. We compared substrate binding of the CYP142A3 enzymes of M. ulcerans and M. marinum and CYP142A1 from M. tuberculosis using UV–vis spectroscopy. The catalytic oxidation of cholesterol derivatives by all three enzymes was undertaken. Both CYP142A3 enzymes were structurally characterized by X-ray crystallography. The amino acid sequences of the CYP142A3 enzymes are more similar to CYP142A1 from M. tuberculosis than CYP142A2 from Mycolicibacterium smegmatis. Both CYP142A3 enzymes have substrate binding properties, which are more resemblant to CYP142A1 than CYP142A2. The cholest-4-en-3-one-bound X-ray crystal structure of both CYP142A3 enzymes were determined at a resolution of <1.8 Å, revealing the substrate binding mode at a high level of detail. The structures of the cholest-4-en-3-one binding CYP142 enzymes from M. ulcerans and M. marinum demonstrate how the steroid binds in the active site of these enzymes. They provide an explanation for the high selectivity of the enzyme for terminal methyl C–H bond oxidation to form 26-hydroxy derivatives. These enzymes in pathogenic Mycobacterium species are candidates for inhibition. The work here demonstrates that similar drug molecules could target these CYP142 enzymes from different species in order to combat Buruli ulcer or tuberculosis.

The bacterial cytochrome P450 (CYP) CYP125 enzymes can competitively oxidise sitosterol in the presence of cholesterol

“…The reactions were analysed by GC-MS after derivization. 34 All three CYP125 enzymes in this study were able to oxidise sitosterol (Fig. 3 and Fig.…”

“…31 Docking studies have proposed that sitosterol is an inhibitor of CYP125, though this argument is based on molecular dynamics simulations. 32 Importantly, Mtb and other bacteria also have genes encoding other P450 enzyme families that can play the same roles in cholesterol/cholest-4-en-3-one oxidation, namely the CYP142 33,34 and CYP124 35 enzyme families. These families also display additional functionality to CYP125.…”

“…S2, ESI †), in contrast to the obvious larger shifts with cholesterol. [33][34][35] We next assessed the activity of these CYP125 enzymes for sitosterol oxidation using a reconstituted NADPH and spinach ferredoxin/ferredoxin reductase electron transfer system. The reactions were analysed by GC-MS after derivization.…”