Thiols, thiol-transferases and related proteins are essential to virtually all aspects of biology. In mammals and microbes thiols maintain redox status, protect cells against toxic electrophiles and participate in crucial biosynthetic, metaboli and catabolic processes. The general objectives of this renewal application address specific problems in the understanding of the involvement of thiol transferases and related proteins in mammalian and microbial biology.
The specific aims are (i) to advance our understanding of the mechanisms and structures of three MAPEG proteins (Membrane Associated Proteins in Eicosinoid and Glutathione metabolism) and (ii) to provide previously inaccessible scientific insight into the chemistry and biology of bacillithiol (BSH);a newly discovered thiol found in Gram-positive microorganisms. Members of the MAPEG superfamily are involved in several aspects of the regulation of pain, fever, inflammation and allergic response in humans. The first specific aim focuses on crucial issues regarding three different MAPEG proteins, microsomal prostaglandin E synthase 1 (MPEGS1), 5-lipoxigenase activating protein (FLAP) and microsomal glutathione transferase 1 (MGST1). All of these proteins are integral membrane proteins.
Specific Aim 1 relies heavily on hydrogen/deuterium exchange mass spectrometry (H/DEX MS) to probe the structural aspects of these proteins as they interact with substrates, inhibitors and protein partners.
Specific Aim 1 a will ascertain if it is possible to perform H/DEX MS on an integral membrane protein MGST1 in its native membrane environment, the endoplasmic reticulum.
Specific Aim 1 b will address the potential inhibition of MPGES1 by nitrosylation of C59 and the chemical mechanism of MPGES1, about which virtually nothing is known.
Specific Aim 1 c is will elucidate the structural details of the interaction of FLAP with arachidonic acid and its interaction with 5-lipoxigenase (5-LOX) in the initiation of the lipoxygenase pathway for the synthesis of leukotrienes.
Specific aim 2 is directed at understanding the role of bacillithiol (BSH) in the resistance of Gram-positive pathogenic microorganisms to the antibiotic fosfomycin conferred by the enzyme FosB.
This aim i s leveraged by the fact that we have, in the last project period with help of the Vanderbilt Institut of Chemical Biology Synthesis Core, synthesized 1.3 grams bacillithiol and have obtained the genes, expressed and purified the FosB enzymes from several sources including Bacillus cereus, Bacillus anthracis and Staphylococcus aureus which puts us in an excellent position to accomplish this aim.
Specific Aim 2 a is to determine the crystal structure of one or more FosB proteins with fosfomycin, BSH or product bound.
Specific Aim 2 b will elucidate the chemistry, kinetics, and divalent metal ion-dependence of the FosB enzymes with both BSH and the alternative substrate L-Cys.
Thiols, thiol-transferases and associated proteins are crucial in the regulation of mammalian and microbial biology related to human health. Their roles in leukotriene and prostaglandin biosynthesis are key to the control of pain, inflammation and allergic responses. Their biological diversity also extends to poorly understood aspects of the resistance of pathogenic microorganisms to antibiotics.
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