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J. Biol. Chem., Vol. 279, Issue 50, 52346-52352, December 10, 2004

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TetX Is a Flavin-dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics^*

Wangrong Yang, Ian F. Moore, Kalinka P. Koteva, David C. Bareich, Donald W. Hughes, and Gerard D. Wright¶

From the Antimicrobial Research Center, Department of Biochemistry and Biomedical Sciences and the Department of Chemistry, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

The tetracycline antibiotics block microbial translation and constitute an important group of antimicrobial agents that find broad clinical utility. Resistance to this class of antibiotics is primarily the result of active efflux or ribosomal protection; however, a novel mechanism of resistance has been reported to be oxygen-dependent destruction of the drugs catalyzed by the enzyme TetX. Paradoxically, the tetX genes have been identified on transposable elements found in anaerobic bacteria of the genus Bacteroides. Overexpression of recombinant TetX in Escherichia coli followed by protein purification revealed a stoichiometric complex with flavin adenine dinucleotide. Reconstitution of in vitro enzyme activity demonstrated a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products of TetX activity were unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions supported initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products. TetX is therefore a FAD-dependent monooxygenase. The enzyme not only catalyzed efficient degradation of a broad range of tetracycline analogues but also conferred resistance to these antibiotics in vivo. This is the first molecular characterization of an antibiotic-inactivating monooxygenase, the origins of which may lie in environmental bacteria.

Received for publication, August 19, 2004 , and in revised form, September 23, 2004.

This article is dedicated to Professor Christopher Walsh on the occasion of his 60th birthday.

^* This work was supported by the Natural Sciences and Engineering Council of Canada and by a Canada Research Chair in Antibiotic Biochemistry (to G. D. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains 20 supplementary figures and 5 tables.

^¶ To whom correspondence should be addressed. Tel.: 905-525-9140 (ext. 22454); Fax: 905-522-9033; E-mail: wrightge{at}mcmaster.ca.

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