Tetracycline is a very important antibiotic that is not only extremely effective, but readily available and therefore used extensively in agricultural and clinical settings. It binds to the 30S ribosomal subunit preventing amino acyl tRNA from binding to the A site of the ribosome completely preventing translation. There are three types of resistance mechanisms prokaryotes have evolved to combat the detrimental effects of tetracycline. The most common is efflux, efflux pumps allow microorganisms to regulate their internal environment by removing toxic substances. Another mechanism is ribosomal protection whereby auxiliary proteins are produced to interact with the ribosome and prevent tetracycline binding. The third mechanism, which is not yet fully understood, is enzymatic degradation of tetracycline by recently evolved enzymes produced by bacterium in response to wide-spread use of tetracycline. Tetracycline monooxygenase (TetX) is one of the enzymes involved in this process. It catalyzes the conversion of tetracycline to 11a- hydrotetracycline that is completely unstable at physiological pH and rapidly decomposes before the drug can bind to the ribosome. The long-term goals of the work carried out this summer is to develop a detailed understanding of the chemical mechanism of TetX which is currently unknown. Knowledge of such a mechanism will enable the design of mechanism-based inhibitors of TetX which will abolish its activity and can be used in conjugation with tetracycline to treat bacterial infection. To this end a kinetic assay was developed this summer that will be used in the coming years in the Francis lab. The enzyme was expressed purified and its steady state kinetic parameters were determined with tetracycline as substrate.

Faculty Mentor: Dr. Kevin Francis

Department of Chemistry