title: Substrate and reaction specificity of Mycobacterium tuberculosis cytochrome P450 CYP121: insights from biochemical studies and crystal structures.
abstract: Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn(85), Phe(168), and Trp(182). The propensity of the cYY tyrosyl to point toward Arg(386) was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.
authors.affiliation: Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette Cedex, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Fonvielle
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Le Du
authors.forename: Olivier
authors.initials: O
authors.lastname: Lequin
authors.forename: Alain
authors.initials: A
authors.lastname: Lecoq
authors.forename: Mickaël
authors.initials: M
authors.lastname: Jacquet
authors.forename: Robert
authors.initials: R
authors.lastname: Thai
authors.forename: Steven
authors.initials: S
authors.lastname: Dubois
authors.forename: Guillaume
authors.initials: G
authors.lastname: Grach
authors.forename: Muriel
authors.initials: M
authors.lastname: Gondry
authors.forename: Pascal
authors.initials: P
authors.lastname: Belin
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010456:Peptides, Cyclic / Q000737:chemistry*; D011485:Protein Binding; D012996:Solutions; D013379:Substrate Specificity
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004151:Dipeptides; D004791:Enzyme Inhibitors; D010456:Peptides, Cyclic; D012996:Solutions; C464825:cytochrome P-450 CYP121; C086820:cyclo(tyrosyl-tyrosyl); D003577:Cytochrome P-450 Enzyme System
keywords: Crystallography; Cytochrome P450; Enzyme Specificity; Enzyme Structure; Nuclear Magnetic Resonance; Tuberculosis