Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis.
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 nm. 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.
McLean
Kirsty J
KJ
Department of Biochemistry, The Adrian Building, University of Leicester, UK.
Cheesman
Myles R
MR
Rivers
Stuart L
SL
Richmond
Alison
A
Leys
David
D
Chapman
Stephen K
SK
Reid
Graeme A
GA
Price
Nicholas C
NC
Kelly
Sharon M
SM
Clarkson
John
J
Smith
W Ewen
WE
Munro
Andrew W
AW
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D017931:DNA Primers; D011994:Recombinant Proteins; C464825:cytochrome P-450 CYP121; D003577:Cytochrome P-450 Enzyme System