Abstract
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.
Highlights
Tetracyclines represent one of the most successful classes of antibiotics used in the past 50 years
Since the first identification of chlortetracycline in 1948 from extracts of Streptomyces aureofaciens, numerous analogues, both natural and semisynthetic, have found clinical use (Fig. 1). This class of antibiotic has been a mainstay in the treatment of bacterial infections, being highly prized for a broad spectrum of antimicrobial activity, oral availability, and low cost
The tetX gene was identified in transposons Tn4351 [10] and Tn4400 [9] harbored by the obligate anaerobe Bacteroides fragilis. Transfer of this gene to aerobically growing Escherichia coli uncovered a cryptic tetracycline resistance activity that was associated with destruction of the antibiotic and a commensurate darkening of the growth medium [9, 10]
Summary
Reagents—Tetracyclines, NADPϩ, NADPH, and glucose-6-phosphate were from Sigma. Glucose-6-phosphate dehydrogenase was from Roche Diagnostics. Inactivation reactions contained 3 mM oxytetracycline, 1 mM NADPϩ, 40 mM glucose-6-phosphate, 0.3 unit of glucose-6-phosphate dehydrogenase, and 10 g of purified TetX2, 25 mM TAPS, pH 8.5, in a total volume of 0.1 ml. The column was equilibrated with H2O plus 0.05% trifluoroacetic acid and tetracyclines, and the products of inactivation were eluted using a linear gradient to 95% CH3CN plus 0.05% trifluoroacetic acid over 14 min at a flow rate of 1 ml/min. NMR Analysis of Oxytetracycline and Inactivation Products—A solution of 4 mM NADPϩ, 40 mM glucose-6-phosphate, and 20 units of glucose-6-phosphate dehydrogenase in 25 mM TAPS, pH 8.5, was incubated at 37 °C for 20 min to generate NADPH. Green at the McMaster Regional Center for Mass Spectrometry on a Waters-Micromass Global Ultima Quadrupole time of flight mass spectrometer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
