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Antimicrobial Agents and Chemotherapy, March 2009, p. 1157-1164, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01237-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification, Characterization, and Azole-Binding Properties of Mycobacterium smegmatis CYP164A2, a Homolog of ML2088, the Sole Cytochrome P450 Gene of Mycobacterium leprae{triangledown}

Andrew G. S. Warrilow,1 Colin J. Jackson,1 Josie E. Parker,1 Timothy H. Marczylo,2 Diane E. Kelly,1 David C. Lamb,1 and Steven L. Kelly1*

Institute of Life Science, Swansea University, Swansea, Wales, United Kingdom,1 Cancer Biomarkers and Prevention Group, RKCSB, Leicester Royal Infirmary, Leicester, United Kingdom2

Received 17 September 2008/ Returned for modification 18 October 2008/ Accepted 7 December 2008

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 (Kd, 1.2 to 2.5 µM); 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.

* Corresponding author. Mailing address: Institute of Life Science, Swansea University, Swansea SA2 8PP, Wales, United Kingdom. Phone: 44 1792 292207. Fax: 44 1792 503430. E-mail: s.l.kelly{at}swansea.ac.uk

{triangledown} Published ahead of print on 15 December 2008.

Antimicrobial Agents and Chemotherapy, March 2009, p. 1157-1164, Vol. 53, No. 3
0066-4804/09/$08.00+0     doi:10.1128/AAC.01237-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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