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Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Hung-wen Liu. Vita. Includes bibliographical references.

Thesis (M.S. in Biochemistry)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

The focus of this dissertation is the study of two enzymes, DXR and HppE. DXR catalyzes the first committed step in the MEP pathway, which is the pathway most eubacteria, archeabacteria, algae, and the plastids of plants use for the biosynthesis of isoprenoid. Since mammals utilize the mevalonate pathway and isoprenoids are essential for survival, all enzymes in the MEP pathway are excellent antibiotic targets. One antibiotic that has promise in the fight against malaria is the natural product fosmidomycin, whose antibiotic activity is due to its ability to bind and inhibit DXR. With a deeper understanding of DXR's catalyzed reaction, it will be possible to design a more sophisticated and potent antibiotic. To probe the mechanism of DXR, two fluorinated substrate analogues, 3F-DXP and 4F-DXP, and a fluorinated product analogue, FCH₂-MEP were designed and analyzed as possible substrates or inhibitors. To further analyze the mechanism of DXR, a 2° [²H]-KIE study was conducted using the equilibrium perturbation method. The second enzyme this dissertation examines is HppE, which catalyzes the final step in the biosynthesis of the antibiotic, fosfomycin. Fosfomycin is a clinically useful antibiotic for the treatment of limb-threatening diabetic foot infections and urinary tract infections. Chemically speaking, HppE is unique for two reasons. First, HppE's epoxidation differs from Nature's standard method of epoxide formation by alkene oxidation, where the epoxide oxygen is derived from molecular oxygen. For HppE, the epoxide is formed through the dehydrogenation of a secondary alcohol; thus the epoxide oxygen is derived from the substrate. Second, HppE is a unique member of the mononuclear non-heme iron-dependent family of enzymes. HppE differs from all other mononuclear non-heme iron-dependent enzymes by requiring NADH and an external electron mediator for turnover but not requiring [alpha]-KG, pterin, ascorbate, or an internal iron-sulfur cluster. After a study was published on the activity of zinc-reconstituted HppE from Streptomyces wedmorensis, the proposed iron and NADH dependent mechanism of HppE was reevaluated and was reconfirmed. The HppE from Pseudomonas syringae (Ps-HppE) was also purified and was characterized biochemically and spectroscopically. The results of [²H] and [¹⁸O]-KIE studies on Ps-HppE are also reported. / text

The pathogen C. jejuni is now recognised as the leading cause of bacterial foodborne enteritis in the industrial world. The yearly estimate for Campylobacter infections in the United States alone is 2.4 million people or 1% of the population. Illness caused by C. jejuni is self-limiting, however, some individuals develop complications resulting in autoimmune responses. Despite being a major health burden, the pathogenic process is not fully understood. One aspect of importance is the ability of C. jejuni to adhere to glycosaminoglycans (GAGs), such as heparin. GAGs, sulphated carbohydrates expressed on or in host cells, can serve as receptors for bacterial proteins. In the first study, five heparin-binding proteins of C. jejuni NCTC 11168H were identified. For PEB3 (Cj0289c), this work showed that native wild-type PEB3 and purified recombinant PEB3 produced in E. coli bind heparin. The location of two PEB3 heparin-binding clusters: 62KAKKD65 and 122NKKVRI127, was investigated via site-directed mutagenesis, resulting in impaired heparin-binding. These data suggest GAG-protein-binding may play a role in the pathogenesis of C. jejuni. As well as GAG-binding PEB3 binds 3-PG. Though its exact in vivo role remains unclear, it may act to deliver 3-PG. Scrutiny of the C. jejuni NCTC 11168H genome revealed an uncharacterised gene next to peb3 encoding glpT, or a putative 3-PG transporter. The location of glpT adjacent to peb3 may suggest a related function for the corresponding proteins with PEB3 as the periplasmic binding partner for the transport of 3-PG via GlpT. In this thesis, the roles of peb3 and glpT for two independent phenotypes, 3-PG dependent growth and fosfomycin sensitivity was studied in vitro. The findings indicate glpT has an effect on both, but not peb3. Furthermore, the NCTC 11168H glpT pseudogene, despite containing two frameshift mutations, has the capacity to encode a functional protein. Lastly, the NCTC 11168H peb3/glpT locus was compared with other C. jejuni strains and closely related species C. coli, C. lari and C. upsaliensis genome sequences. The majority of strains peb3/glpT locus followed the gene arrangement lpxB, peb3, glpT, surE. However, the findings indicate the gene loci between lpxB/surE in remaining strains to be hypervariable. Further analysis shows peb3 to be relatively conserved, whereas, the majority of glpT genes display genetic diversity due to interruptions such as indels and deletion. Lastly, I display the organisation of the peb3/glpT locus and glpT structure in their evolutionary context through MLST. In summary, the findings provide for further characterisation of the PEB3 protein and explores the importance of the uncharacterised GlpT of C. jejuni.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

Yes / Bacillus sp. is a Gram-positive bacterium that is commonly found in seawater. In this study, the genome of marine Bacillus sp. strain G3(2015) was sequenced using MiSeq. The fosfomycin resistant gene fosB was identified upon bacterial genome annotation. / University of Malaya through HIR grants (UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027; UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/01, A000001- 50001); Postgraduate Research grant PG083-2015B

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