Characterization of Active Site Structure in CYP121: A CYTOCHROME P450 ESSENTIAL FOR VIABILITY OF MYCOBACTERIUM TUBERCULOSIS H37Rv*S⃞
Other literature type
McLean, Kirsty J.
Lewis, D. Geraint
Dunford, Adrian J.
Seward, Harriet E.
Cheesman, Myles R.
Smith, W. Ewen
Cole, Stewart T.
Munro, Andrew W.
- Publisher: American Society for Biochemistry and Molecular Biology
Protein Structure and Folding
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