One of the major challenges in biochemistry is to understand the functional genomics of organisms. It is a staggering problem when one considers the fact that about 40% of the genes in of one of the best-understood organisms, Escherichia coli, are not experimentally defined. The organism colonizes the human intestine shortly after birth and is, along with its close relatives such as Salmonella, Shigella and Yersinia, a pathogen that is a significant source of human disease. Glutathione (GSH) is the predominant redox active thiol in most aerobic organisms where it plays a fundamental role in metabolic, catabolic and redox chemistry. GSH transferases are enzymes that participate in this chemistry by adding GSH to electrophilic acceptors. The E. coli genome harbors genes encoding nine glutathione (GSH) transferase homologues. Amazingly, only one gene has a reasonably well-defined function and it does NOT encode a GSH transferase but rather stringent starvation protein A, SspA, a transcription factor. Under aerobic conditions, GSH is the predominate thiol in E. coli. However it is largely converted to glutathionylspermidine (GspSH) under anaerobic conditions by glutathionylspermidine synthetase/amidase (GSS). The broad, long-term objectives of this project are to understand the GSH/GspSH homeostasis in E. coli and the biological activities and roles of chromosomally encoded GSH transferase homologues in E. coli. Ten interrelated proteins are the subject of this investigation. They include the eight canonical GSH transferase homologues, YliJ, YncG, Gst, YfcF, YfcG, YghU, SspA, and YibF as well as GSS, and the membrane-bound GSH transferase, YecN. The research plan includes three specific aims.
The first aim i s to understand the mechanism and regulation of GSH/GspSH homeostasis in E. coli.
The second aim i s to define the biological functions of the eight canonical GSH transferase homologues in E. coli.
The third aim i s to determine the biological role of the single membrane-bound GSH transferase homologue.
These aims will be achieved by a multidisciplinary approach that includes: (i) analysis of genome context;(ii) phenotypic responses to gene knockouts;(iii) response of gene expression to growth conditions and stress;(iv) a search for protein partners;(v) determination of X-ray crystal structures of the proteins;(vi) functional and kinetic characterizations of the proteins.
Escherichia coli is a member of the Enterobacteriaceae family that includes several intestinal pathogens such as Salmonella, Shigella and Yersinia. The bacterium typically colonizes the human intestine shortly after birth where it remains the predominant facultative microorganism. Understanding the biochemistry of E. coli is fundamental to understanding its pathogenesis.
|Droege, Kristin D; Keithly, Mary E; Sanders, Charles R et al. (2017) Structural Dynamics of 15-Lipoxygenase-2 via Hydrogen-Deuterium Exchange. Biochemistry 56:5065-5074|
|Spahiu, Linda; Ålander, Johan; Ottosson-Wadlund, Astrid et al. (2017) Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation. Biochemistry 56:3089-3098|
|Whitson, Jeremy A; Sell, David R; Goodman, Michael C et al. (2016) Evidence of Dual Mechanisms of Glutathione Uptake in the Rodent Lens: A Novel Role for Vitreous Humor in Lens Glutathione Homeostasis. Invest Ophthalmol Vis Sci 57:3914-25|
|Winchell, Kelsey R; Egeler, Paul W; VanDuinen, Andrew J et al. (2016) A Structural, Functional, and Computational Analysis of BshA, the First Enzyme in the Bacillithiol Biosynthesis Pathway. Biochemistry 55:4654-65|
|Shen, Jiangchuan; Keithly, Mary E; Armstrong, Richard N et al. (2015) Staphylococcus aureus CstB Is a Novel Multidomain Persulfide Dioxygenase-Sulfurtransferase Involved in Hydrogen Sulfide Detoxification. Biochemistry 54:4542-54|
|VanDuinen, Andrew J; Winchell, Kelsey R; Keithly, Mary E et al. (2015) X-ray crystallographic structure of BshC, a unique enzyme involved in bacillithiol biosynthesis. Biochemistry 54:100-3|
|Thompson, Matthew K; Keithly, Mary E; Goodman, Michael C et al. (2014) Structure and function of the genomically encoded fosfomycin resistance enzyme, FosB, from Staphylococcus aureus. Biochemistry 53:755-65|
|Gentry, Leslie E; Thacker, Matthew A; Doughty, Reece et al. (2013) His86 from the N-terminus of frataxin coordinates iron and is required for Fe-S cluster synthesis. Biochemistry 52:6085-96|
|Thompson, Matthew K; Keithly, Mary E; Harp, Joel et al. (2013) Structural and chemical aspects of resistance to the antibiotic fosfomycin conferred by FosB from Bacillus cereus. Biochemistry 52:7350-62|
|Prage, Edward B; Morgenstern, Ralf; Jakobsson, Per-Johan et al. (2012) Observation of two modes of inhibition of human microsomal prostaglandin E synthase 1 by the cyclopentenone 15-deoxy-?(12,14)-prostaglandin J(2). Biochemistry 51:2348-56|
Showing the most recent 10 out of 72 publications