Modern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and unnatural compounds that are structurally distinct from their target substrate. The widespread existence of such promiscuity could prove generally useful both in the natural and directed evolution of new proteins. To probe the persistence of enzyme promiscuity in modern proteomes we studied the model organism Escherichia coli due to its rapid growth, ease of genetic manipulation and many years of prior research on this organism which have generated abundant knowledge on its metabolism. The first exploration into uncovering enzyme promiscuity, described in chapter two, examines the proton transfer reaction catalyzed by triosephosphate isomerase (TIM). Triosephosphate isomerase catalyzes the interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the E. coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding for a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of L-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate. In chapter three we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a Kd value of 5.8 ± 0.2 µM in the absence of NADPH and a Kd value of 11 ± 1.3 µM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process. In chapter four we probe the network of genes within E. coli that can provide resistance to the nonnatural toxin bromoacetate. Microbial niches contain toxic chemicals that are capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival following exposure to a lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. Genetic experiments also identify 63 single-gene mutants of E. coli that display increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene that encodes a glutathione transferase capable of catalyzing the detoxification of bromoacetate with a kcat/Km value of 5.4 × 103 M-1 s-1. The catalytic proficiency of YliJ, which exceeds 5 orders of magnitude, is particularly noteworthy considering the enzyme is unlikely to have previously encountered bromoacetate. In total, our results indicate that nearly 2% of the E. coli proteome contributes to, or can be recruited to provide, bromoacetate resistance. This illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins. The work described here illuminates the vast metabolic and functional plasticity of protein function harbored within bacteria. Their ability to recruit latent and weakly active proteins for novel functions enables survival under diverse nutritional and environmental challenges. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of
Philosophy. / Summer Semester, 2010. / June 14, 2010. / Enzyme Function, Antibiotic Resistance, Glutathione Transferase / Includes bibliographical references. / Brian Miller, Professor Directing Dissertation; Hank Bass, University Representative; Hong Li, Committee Member; Lei Zhu, Committee Member; M. Elizabeth Stroupe, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_182701 |
| Contributors | Desai, Kevin (authoraut), Miller, Brian (professor directing dissertation), Bass, Hank (university representative), Li, Hong (committee member), Zhu, Lei (committee member), Stroupe, M. Elizabeth (committee member), Department of Chemistry and Biochemistry (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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