title: Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv.
abstract: Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.
authors.affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. Kirsty.McLean@Manchester.ac.uk
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.forename: Paul
authors.initials: P
authors.lastname: Carroll
authors.forename: D Geraint
authors.initials: DG
authors.lastname: Lewis
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Dunford
authors.forename: Harriet E
authors.initials: HE
authors.lastname: Seward
authors.forename: Rajasekhar
authors.initials: R
authors.lastname: Neeli
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.forename: Laurent
authors.initials: L
authors.lastname: Marsollier
authors.forename: Philip
authors.initials: P
authors.lastname: Douglas
authors.forename: W Ewen
authors.initials: WE
authors.lastname: Smith
authors.forename: Ida
authors.initials: I
authors.lastname: Rosenkrands
authors.forename: Stewart T
authors.initials: ST
authors.lastname: Cole
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry*; D001393:Azoles / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain / Q000502:physiology*; D003067:Coenzymes / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005816:Genetic Complementation Test; D006418:Heme / Q000737:chemistry / Q000235:genetics; D007501:Iron / Q000737:chemistry; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D013816:Thermodynamics
chemical_list: D000995:Antitubercular Agents; D001393:Azoles; D001426:Bacterial Proteins; D003067:Coenzymes; D065607:Cytochrome P-450 Enzyme Inhibitors; C464825:cytochrome P-450 CYP121; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D007501:Iron