Expression and characterization of Mycobacterium tuberculosis CYP144: Common themes and lessons learned in the M. tuberculosis P450 enzyme family

Max D. Driscoll, Kirsty J. McLean, Myles R. Cheesman, Thomas A. Jowitt, Marjorie Howard, Paul Carroll, Tanya Parish, Andrew W. Munro

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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 (Kd = 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.

Original languageEnglish
Pages (from-to)76-87
Number of pages12
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1814
Issue number1
Early online date8 Jun 2010
DOIs
Publication statusPublished - 1 Jan 2011
Externally publishedYes

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