Pseudomonas aeruginosa encodes a putative topoisomerase with sequence similarity to the type IB topoisomerase enzyme from vaccinia virus. Residues in the active site are conserved, notably Tyr292 which would be predicted to form the transient covalent bond to DNA. The gene encoding the P. aeruginosa topoisomerase I (PAT) was cloned and expressed in E. coli. The enzyme relaxes supercoiled DNA, while a mutant of P. aeruginosa containing a Tyr292 to Phe substitution at the active site was found to be catalytically inert. This is consistent with the role of Tyr in forming the covalent intermediate. Like vaccinia topoisomerase (VT), PAT relaxes DNA in the absence of ATP. Unlike VT, PAT does not relax supercoiled DNA without MgCl2 (or MnCl2) present. In addition, high concentration of NaCl is not able to substitute for MgCl2 as seen for VT. A truncated derivative of the topoisomerase lacking residues 1-98 relaxes DNA in the absence of the N-terminal domain, with both full length and truncated enzyme exhibiting equivalent requirements for divalent cations. Data shows that P. aeruginosa encodes a functional topoisomerase with significant similarity to the type IB enzyme encoded by poxviruses.
Fluoroquinolones are antibacterial agents in clinical use with activity against DNA gyrase and DNA topoisomerase IV. P. aeruginosa is an opportunistic pathogen causing life-threatening diseases. Sparfloxacin, enrofloxacin and norfloxacin fluoroquinolones, are able to inhibit the PAT at high concentrations but other drugs belonging to this family are unable to do so.
VT is ¡Ö32 KDa and one-third the size of the human topoisomerase ¡Ö 100 KDa. It shares sequence and biochemical similarities with the human topoisomerase. The VT binds duplex DNA with stringent specificity for transesterification at 5`-(C/T) CCTT site, where the 3` phosphate of the incised strand is linked to the Tyr274 of the enzyme to form a covalent cleavage complex. The fluoroquinolone enrofloxacin inhibits relaxation of supercoiled DNA by VT in a Mg2+-dependent fashion. Further results indicate that the mechanism by which enrofloxacin inhibits VT is by preventing formation of the covalent complex which suggest that fluoroquinolones may be structurally optimized to target type IB topoisomerases.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-01202009-104542 |
| Date | 20 January 2009 |
| Creators | Jain, Teesta |
| Contributors | Patrick DiMario, Marcia Newcomer, Craig Hart, Anne Grove, James Ottea |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-01202009-104542/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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