Expression and characterization of Mycobacterium tuberculosis CYP144: common themes and lessons learned in the M. tuberculosis P450 enzyme family.
CYP144 from Mycobacterium tuberculosis was expressed and purified. CYP144 demonstrates heme thiolate coordination in its ferric form, but the cysteinate is protonated to thiol in both the carbon monoxide-bound and ligand-free ferrous forms (forming P420 in the former). Tight binding of various azole drugs was shown, with affinity for miconazole (K(d)=0.98 μM), clotrimazole (0.37 μM) and econazole (0.78 μM) being highest. These azoles are also the trio with the highest affinity for the essential CYP121 and for the cholesterol oxidase CYP125 (essential for host infection), and have high potency as anti-mycobacterial drugs. Construction of a Mtb gene knockout strain demonstrated that CYP144 is not essential for growth in vitro. However the deletion strain was more sensitive to azole inhibition in culture suggesting an important role for CYP144 in cell physiology and/or in mediating azole resistance. The biophysical and genetic features of CYP144 are compared to those of other characterized Mtb P450s, identifying both commonality in properties (including thiolate protonation in ferrous P450s) and intriguing differences in thermodynamic and spectroscopic features. Our developing knowledge of the Mtb P450s has revealed unusual biochemistry and gene essentiality, highlighting their potential as drug targets in this human pathogen.
Driscoll
Max D
MD
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M17DN, UK.
McLean
Kirsty J
KJ
Cheesman
Myles R
MR
Jowitt
Thomas A
TA
Howard
Marjorie
M
Carroll
Paul
P
Parish
Tanya
T
Munro
Andrew W
AW
D000891:Anti-Infective Agents, Local / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D003022:Clotrimazole / Q000378:metabolism / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004464:Econazole / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques; D007700:Kinetics; D008825:Miconazole / Q000378:metabolism; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010084:Oxidation-Reduction; D011199:Potentiometry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013997:Time Factors
D000891:Anti-Infective Agents, Local; D001426:Bacterial Proteins; C556633:CYP144 protein, Mycobacterium tuberculosis; D011994:Recombinant Proteins; D004464:Econazole; D008825:Miconazole; D003577:Cytochrome P-450 Enzyme System; D003022:Clotrimazole