Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis

Kirsty J McLean, Myles R Cheesman, Stuart L Rivers, Alison Richmond, David G Leys, Stephen K Chapman, Graeme A Reid, Nicholas C Price, Sharon M Kelly, John Clarkson, W Ewen Smith, Andrew W Munro

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    Abstract

    The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 run. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen. (C) 2002 Elsevier Science Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)527-541
    Number of pages15
    JournalJournal of Inorganic Biochemistry
    Volume91
    Issue number4
    DOIs
    Publication statusPublished - 20 Sep 2002
    Event12th International Conference on Advances in the Inorganic Biochemistry of Cytochrome P450 - La Grande Motte, France
    Duration: 11 Sep 200115 Sep 2001

    Keywords

    • NUCLEOTIDE-SEQUENCE
    • CIRCULAR-DICHROISM
    • azole inhibitors
    • NITRIC-OXIDE SYNTHASE
    • BACILLUS-SUBTILIS
    • STEROL 14-ALPHA-DEMETHYLASE
    • ACTIVE-SITE
    • ELECTRON-PARAMAGNETIC-RESONANCE
    • CRYSTAL-STRUCTURE
    • COMPLETE GENOME SEQUENCE
    • CYP121
    • P450
    • drug targets
    • Mycobacterium tuberculosis

    Cite this

    McLean, K. J., Cheesman, M. R., Rivers, S. L., Richmond, A., Leys, D. G., Chapman, S. K., Reid, G. A., Price, N. C., Kelly, S. M., Clarkson, J., Smith, W. E., & Munro, A. W. (2002). Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis. Journal of Inorganic Biochemistry, 91(4), 527-541. https://doi.org/10.1016/S0162-0134(02)00479-8