Expression, purification and spectroscopic characterization of the cytochrome P450CYP121 from Mycobacterium tuberculosis

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

83 Citations (Scopus)

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
Article numberPII S0162-0134(02)00479-8
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: 1 Sep 2001 → …

Keywords

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

Fingerprint

Dive into the research topics of 'Expression, purification and spectroscopic characterization of the cytochrome P450CYP121 from Mycobacterium tuberculosis'. Together they form a unique fingerprint.

Cite this