Resistance to the tetracycline antibiotics occurs primarily by efflux and ribosome protection mechanisms, however a tetracycline inactivating enzyme, TetX, was identified 15 years ago, although little is known about this mechanism. The gene encoding this enzyme was identified on a ππ’π€π΅π¦π³π°πͺπ₯π¦π΄ transposon and results from DNA sequence analysis and studies of bacterial culture media suggested that π΅π¦π΅π's protein product might be a NADPH-requiring oxidase (ππ±π¦π¦π³ ππ π’π―π₯ ππ’ππΊπ¦π³π΄ ππ. π ππ’π€π΅π¦π³πͺπ’π. 171(1): 148-153, 1989). We have expressed a copy of π΅π¦π΅πΉ gene, π΅π¦π΅πΉ2, in Escherichia coli, and purified the enzyme to high purity. We showed that TetX2 is a monomeric 44 kDa cytoplasmic protein and UV-Vis and HPLC studies established that TetX contained an FAD cofactor. Continuous and stopped enzyme assays have been developed and established that that the enzyme requires 0β and NADPH for tetracycline degradation. Liquid chromatographic mass spectrometry (LC -MS) analysis of TetX reaction products using oxytetracycline (461 Da) as a substrate indicated that the enzyme catalyses the incorporation of one oxygen atom into oxytetracycline, resulting in a compound of 477 Da with no antibiotic activity. Steady state kinetic analysis demonstrated that TetX2 has a broad substrate specificity with the capacity to inactivate several members of the tetracycline family tested. Identification of the inactivated tetracycline product revealed that the tetracycline inactivation process is a TetX2 catalyzes tetracycline oxidation reaction. These studies provide the first biochemical analysis of a tetracycline inactivating enzyme. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22730 |
| Date | 12 1900 |
| Creators | Yang, Wangrong |
| Contributors | Wright, G. D., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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