Characterization of Active Site Structure in CYP121

• Published in: The Journal of biological chemistry Vol. 283; no. 48; p. 33406
• Main Authors: Kirsty J. McLean, Paul Carroll, D. Geraint Lewis, Adrian J. Dunford, Harriet E. Seward, Rajasekhar Neeli, Myles R. Cheesman, Laurent Marsollier, Philip Douglas, W. Ewen Smith, Ida Rosenkrands, Stewart T. Cole, David Leys, Tanya Parish, Andrew W. Munro
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 28.11.2008
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M802115200

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 K d 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. Pro 346 was shown to control heme cofactor conformation, whereas Arg 386 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.