title: Identification, characterization, and azole-binding properties of Mycobacterium smegmatis CYP164A2, a homolog of ML2088, the sole cytochrome P450 gene of Mycobacterium leprae.
abstract: The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (K(d), 1.2 to 2.5 muM); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.
authors.affiliation: Institute of Life Science, Swansea University, Swansea, Wales, United Kingdom.
authors.forename: Andrew G S
authors.initials: AG
authors.lastname: Warrilow
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Jackson
authors.forename: Josie E
authors.initials: JE
authors.lastname: Parker
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Marczylo
authors.forename: Diane E
authors.initials: DE
authors.lastname: Kelly
authors.forename: David C
authors.initials: DC
authors.lastname: Lamb
authors.forename: Steven L
authors.initials: SL
authors.lastname: Kelly
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001393:Azoles / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009166:Mycobacterium leprae / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D001393:Azoles; D011994:Recombinant Proteins; D012965:Sodium Chloride; D003577:Cytochrome P-450 Enzyme System