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

DOI: 10.1074/jbc.M116.748822 DOI Help

Authors: Jude T. Chenge (University of Manchester) , Le Van Duyet (University of Manchester) , Shalini Swami (University of Manchester) , Kirsty J. Mclean (University of Manchester) , Madeline E. Kavanagh (University of Cambridge) , Anthony G. Coyne (University of Cambridge) , Stephen E. J. Rigby (University of Manchester) , Myles R. Cheesman (University of East Anglia) , Hazel M. Girvan (University of Manchester) , Colin W. Levy (University of Manchester) , Bernd Rupp (Leibniz-Institut für Molekulare Pharmakologie) , Jens P. Von Kries (Leibniz-Institut für Molekulare Pharmakologie) , Chris Abell (University of Cambridge) , David Leys (University of Manchester) , Andrew W. Munro (University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 292 , PAGES 1310 - 1329

State: Published (Approved)
Published: January 2017
Diamond Proposal Number(s): 311 , 1224 , 7146

Open Access Open Access

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.

Journal Keywords: cytochrome P450; electron paramagnetic resonance (EPR); enzyme structure; high throughput screening (HTS); mass spectrometry (MS); Mycobacterium tuberculosis; CYP126A1; redox potentiometry

Subject Areas: Biology and Bio-materials
Collaborations: Diamond Manchester

Instruments: I03-Macromolecular Crystallography

Documents:
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