Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1

Abstract

The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands “moonlight” as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

Working at the membrane interface: Ligand‐induced changes in dynamic conformation and oligomeric structure in human aromatase

“…Thus, anastrozole is likely to promote a conformational change that shifts the equilibrium toward the formation of monomers. Such a behavior has been recently reported for CYP126A1, where an azole‐containing inhibitor (ketoconazole) was found to cause dissociation of the P450 dimer .…”

Advances in the Understanding of Protein-Protein Interactions in Drug Metabolizing Enzymes through the Use of Biophysical Techniques

“…In this aspect, X-ray crystallography has contributed to our structural understanding of the factors involved in dimer formation. For example, the M. tuberculosis CYP126A1 protein was recently demonstrated to form homodimers under crystallization conditions (Chenge et al, 2017 ). The dimer interface is composed of interactions along the hydrophobic B/C and F/G loop regions of the protein, which are known substrate recognition regions.…”

“…Interestingly, the CYP126A1 protein forms a dimer both in the ligand-free state and also with substrate bound. However, the dimer is disrupted when the inhibitor ketoconazole is bound to the protein, leading to conversion to the monomer (Chenge et al, 2017 ). Similarly, CYP2C8 has been shown to crystalize as a dimer (Schoch et al, 2008 ).…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…Interestingly, the CYP126A1 protein forms a dimer both in the ligand-free state and also with substrate bound. However, the dimer is disrupted when the inhibitor ketoconazole is bound to the protein, leading to conversion to the monomer (Chenge et al, 2017). Similarly, CYP2C8 has been shown to crystalize as a dimer (Schoch et al, 2008).…”

“…In this aspect, X-ray crystallography has contributed to our structural understanding of the factors involved in dimer formation. For example, the M. tuberculosis CYP126A1 protein was recently demonstrated to form homodimers under crystallization conditions (Chenge et al, 2017). The dimer interface is composed of interactions along the hydrophobic B/C and F/G loop regions of the protein, which are known substrate recognition regions.…”