The low molecular weight (L.M.W.) thiol redox couple formed by γ-L-glutamyl-L-cysteinyl glycine, also called glutathione (reduced and oxidized), is present in most eukaryotes and a few species of bacteria. Glutathione plays a role in numerous cellular processes by providing a means of shuttling electrons to different enzymatic systems. As a result, thiol-dependent redox metabolic processes are highly coupled. Due to tight coupling of redox reactions, it is difficult to understand how changes in the concentration of glutathione would affect a specific glutathione-dependent process. Interestingly, only a small subset of bacteria encode the canonical enzyme for the biosynthesis of glutathione, namely γ-glutamyl cysteine synthetase (gshA gene product). The mechanisms by which glutathione-dependent processes are carried out in bacteria which do not have the genes for biosynthesis of glutathione or other L.M.W. thiols is not well understood. A genetic selection to restore a glutathione-dependent phenotype in E. coli, lacking the gene involved in first step of glutathione biosynthesis (gshA), was used to address how bacteria lacking gshA might substitute for glutathione. Genetic and biochemical analyses of the E. coli mutants isolated in the selection revealed a de novo pathway for biosynthesis of γ-glutamyl cysteine, the product formed normally by GshA. Additionally we found that the unnatural analogue of glutathione, γ-glutamyl homocysteine could also be formed by this pathway. Bioinformatic analysis suggested that bacteria lacking gshA may use these de novo pathways for biosynthesis of γ-glutamyl cysteine or γ-glutamyl homocysteine, which could serve as potential substitutes for glutathione. The engineering of de novo biosynthetic pathways for γ-glutamyl cysteine and γ-glutamyl homocysteine provided us a strategy for engineering a pathway for biosynthesis of another unnatural analogue of glutathione, β-aspartyl cysteine. Both γ-glutamyl homocysteine and β-aspartyl cysteine could potentially be used as orthologus redox couples in E. coli operating in parallel to glutathione to shuttle electrons to specific pathways which may thus be decoupled from glutathione availability. Glutathione-dependent enzymes that can use orthologous redox couples instead are biochemically isolated from network of other redox reactions in the cell and could be used to direct metabolic fluxes to specific pathways with high efficiencies. Towards this end, we show that glutathione transferase, a glutathione-dependent enzyme, can be engineered to use analogous thiols like γ-glutamyl cysteine as cofactors. / text

No description available.

Multidrug resistance protein (MRP)1, a member of the ABCC branch of the ATP-binding cassette (ABC) superfamily of transporters, can confer resistance to a broad spectrum of chemotherapeutic agents. In addition to the core functional unit of ABC transporters that consists of two membrane spanning domains (MSD) and two nucleotide binding domains (NBD), MRP1 contains a third MSD (MSD0) resulting in the following arrangement: NH2-MSD-(MSD-NBD)2. In lieu of high-resolution structural information for MRP1, cysteine scanning mutagenesis (CSM) was applied to MRP1 and involves the development of a functional template devoid of cysteines into which paired cysteines can be introduced. Previous attempts to create a functional, cys-less template of MRP1 demonstrated that cysteines in MSD0 were structurally and functional important (1;2). However, given that MRP1 lacking MSD0 remains functional, a partially functional, cys-less MRP1 lacking this domain has been expressed in yeast (3-5). Given these results, with the ultimate goal of applying CSM to MRP1 in its entirety, we investigated the endogenous cysteines within MSD0 and co-expressed MRP1 half-molecules and validated these potential CSM templates by transport and ATP binding/hydrolysis assays. Mutation of cysteines within the core of MRP1 had detrimental effects on MRP1 transport activity and further mutation of cysteines by domain revealed that wild-type activity was retained in an MSD0-less MRP1 dual lacking cysteines in both NBDs. This construct was used for introduction of cysteines on juxtaposed faces of the NBD1:NBD2 heterodimer at positions 775 and 1329; comparable residues in the related Cystic Fibrosis Transmembrane Regulator (CFTR/ABCC7) have been suggested to be evolutionarily coupled and joined by a hydrogen bond, maintained in structures of related proteins (6). Unfortunately, functional assays revealed that introduction of cysteines at these positions greatly reduced transport activity of MRP1 and diminished trapping of nucleotide at both NBDs. Finally, alanine substitution of the seven cysteines in MSD0 was not without effect and cellular trafficking assays, co-expression studies and SDS-PAGE analysis suggested an altered conformation of this domain. In addition, a disulfide pair of Cys7 and Cys32 was suggested by these experiments in MSD0 and further supported by examination of these mutants in full-length MRP1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-02-17 12:51:38.13

No description available.

Proefschrift Maastricht. / Lit. opg. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph.D.)--Baylor University, 2008. / Includes bibliographical references (p. 296-303).

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2004. / Dr. Suzanne Shuker, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Donald Doyle, Committee Member ; Dr. Nicholas Hud, Committee Member ; Dr. James C. Powers, Committee Chair. Includes bibliographical references.