Characterisation of the doxorubicin pump of Streptomyces peucetius. The DrrA component of the DrrAB ATP-binding cassette transporter

Zamora G., Rafael E (2006) Characterisation of the doxorubicin pump of Streptomyces peucetius. The DrrA component of the DrrAB ATP-binding cassette transporter. PhD thesis, University of Glasgow.

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Abstract

The emergence of antibiotic resistant microorganisms is one of the most concerning problems in health welfare, and particularly, that of multi drug resistance. The active efflux of compounds from the cell is one of the key strategies used by microorganisms to avoid the noxious effects of toxic compounds and has become one of the most important mechanisms of multiple resistance, such transport processes being catalysed by an array of membrane associated proteins. ATP-Binding Cassette Transporters, that carry out an energy dependent active transport process by which substances cross the cell membrane on the hydrolysis of ATP, also display the ability to translocate a number of unrelated molecules, including antibiotics. These transporters have been identified as one of the largest and most widely distributed families of such transmembrane transport systems. Virtually ubiquitous in nature, ABC transporters have been found in the genomes of every organism from the simplest archea through to man. The fact that they contribute the main pathway for resistance to anticancer drugs in humans highlights their importance and urgent need to study them. In addition to the well known groups of antibiotics produced by members of the Streptomyces genus, that include aminoglycosides, tetracyclines, chloramphenicol, and some (3-lactams, which inhibit the protein synthesis or the proper formation of bacterial cell walls, the species Streptomyces peucetius produces the antibiotics doxorubicin and daunorubicin. These latter compounds are classified into the anthracyclines group and possess antitumoral activity, expanding the arsenal of compounds with different activity produced by Streptomyces. The manner, in which S. peucetius avoids the effect of the antitumorals it produces, is by pumping them out of the cells, and the system that it utilises is that of an ABC transporter. In this system two subunits are present, one of them, DrrA, a peripheral membrane protein that acts as the energy-transducing component, and the other, DrrB, the membrane carrier. This type of permease carries out export of antibiotics in an ATP-dependent manner. The expression of DrrA in E. coli proved to be a challenging enterprise as only low yields were obtained for DrrA at 16 °C, that were only improved when DrrA was fused to Thioredoxin. The over expression and purification of DrrA allowed a partial characterisation of the catalytic activity of DrrA fused to Thioredoxin, with traditional biochemical methods, complemented by additional characterisation assays in wild type DrrA. The characterisation was based on assays of the ATPase activity of Thio- DrrA and DrrA, which displayed a measurable catalytic activity compatible with a role in energising transmembrane transport. These proteins were shown to be cation dependent ATPases able of binding and hydrolysing ATP in a similar manner to the NBD proteins of well-characterised prokaryotic ABC transporters, also showing common features to other NBD's.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Robert Aitken
Keywords: Microbiology
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71048
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71048

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