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The D-glucose disaccharide α, α-D-trehalose is synthesized by a variety of bacteria, fungi, plants and invertebrates to support cell survival by functioning as a fuel, a metabolic regulator or a protectant against environmental stress. Five different trehalose biosynthetic pathways are known to exist, one of which, the OtsA/OtsB pathway is common among pathogenic bacteria and fungi and is also found in parasitic nematodes. Previously reported otsA and otsB gene knockout (or knockdown) experiments have shown that both pathway enzymes are essential for M. tuberculosis cell growth and host colonization. RNAi gene silencing carried out in the nematode model system Caenohabditis elegans revealed that the T6P phosphatase is essential. Based on these findings we concluded that trehalose-synthesizing pathogens are likely to be vulnerable to the action of small molecule inhibitors of T6P phosphatase. We designed a bi-module inhibitor prototype. Accordingly, the phosphate group of the trehalose 6-phosphate moiety was replaced by a phosphate mimetic for targeting the active site of the catalytic domain, and the glucose unit was modified for targeting the cap domain for induced cap closure over the active site. Sulfate proved to be the most effective warhead for the substrate-binding site. T6S binding was shown by using Single Angle X-ray Scattering (SAXS) analysis to induce cap closure. Using glucose-6-sulfate as the platform, a series of synthetic derivatives possessing 'drug-like' functions tethered to the glucose ring were evaluated. Of these first generation inhibitors 4-n-octylphenyl-α-D-glucopyranoside 6-sulfate proved to be the tightest binding T6PP competitive inhibitor. In parallel, we have used the glucose-6-sulfate as the scaffold in the design of active site-directed, irreversible inhibitors. From the adducts which were synthesized and tested for T6PP inactivation, 4-n-octylphenyl-2-(3-(flurosulfonyl) benzoylamino)-2-deoxy-α-D-glucopyranoside-6-sulfate was selec

For my doctoral work, I have developed strategies to mine public databases for data that can be used to infer structural and functional information for the hotdog-fold and HADSF superfamilies. For the hotdog-fold superfamily, I used curated and automatically applied annotations of structure, taxonomic lineage, function, and subfamily membership from the UniProtKB, gene context and taxonomic information from the NCBI, and the results of several in-depth explorations of subfamily/function and structural class membership. Based on the distribution of the aforementioned annotations mapped onto a sequence similarity network (SSN), I applied structural assignments to sequences and/or specific function/subfamily assignments to ~143,000 sequences and general subfamily assignments to an additional ~61,000 sequences. I also identified 52 clusters containing nearly 9,000 uncharacterized sequences lacking any annotations whatsoever and several probable instances of cross-domain gene transfer that would be of interest for further study. Within the thioesterase family of the hotdog-fold superfamily, I identified ~450 targets to undergo high-throughput screens in Karen Allens lab, the SSN-mapped results of which underscore widespread promiscuity across the family. I demonstrated the use of HTS and gene context results to infer functional identities for hotdog-fold superfamily members, though most gene contexts proved to be unilluminating. In the HADSF, I explored the diversity and function space of Firmicutes members, revealing the wide range of HADSF representatives even within members of the same genus. SSNs mapped according to taxonomic lineage, subfamily membership, and function revealed several instances of probable gene transfer among Firmicutes members, but also across phyla. Related gene context, biological range, and HTS results revealed a member of Listeria innocua to be a member of the PTS pathway and provided potentially useful information for other HADSF members. T

Some of the biggest contributors to cellular respiration (and cellular metabolism in general) are acyl-CoA derivatives, a subclass of biological thioesters. Known to function in a variety of pathways, the regulation of their formation and breakdown are critical, carried about by acyl-CoA synthetases and thioesterases, respectively. The work reported within this dissertation will focus on functional divergence within two enzyme superfamilies -- the hot dog-fold and acyl-adenylate-forming superfamilies — and can be broken down into two main parts. Part one will look at tracking the functional divergence within the hot dog-fold superfamily thioesterases. A highly evolved thioesterase, flK, has been found to function in the critical, and highly specific role of fluoroacetate detoxification within the fluorometabolite-producing bacteria, S. cattleya. Using an extensive bioinformatics analysis, flK orthologs were identified and tracked throughout all three domains of life, primarily in bacteria that make up the gut microbiome. Additionally, sequence and structural analyses revealed distinct flK scaffolds, a further indication of divergent functionality. Various flK orthologs were then isolated, cloned and subjected to substrate screening by measuring their individual steady-state kinetic parameters kcat, Km and kcat/Km. Combined with gene context analyses, divergent in vivo functionality was assigned to members of the flK subfamily, as they were proposed to be involved in supplying formate for the one-carbon pool. Part two will focus on the functional characterization of the acyl-CoA synthetases (ligases) in Pseudomonas aeruginosa, the dominant pathogen present in all patients with cystic fibrosis and the leading cause of morbidity and mortality within this afflicted population. Nine freestanding ligases were cloned, isolated and subjected to an extensive substrate screening for acyl-CoA synthetase activity using a novel high-throughput assay. Individual activities wer