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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

The enzymes that hydrolyze thioesters to the free acids and free thiols are called thioesterases. Based on the different folding strategy, most thioesterases are classified into two superfamiles: the hotdog-fold enzyme superfamily or alpha/beta-fold hydrolase enzyme superfamily. Hotdog-fold thioesterases share high degree similarity in structure which resembles a hotdog'. YigI is one of the last two uncharacterized hotdog-fold thioesterases from E.coli. To study the function of YigI, substrate specificity profile was determined by thioesterase activity assay. YigI possesses a preference for long chain acyl-CoA over shorter chain acyl-CoA. The highest activity was found in catalysis of myristoyl-CoA. By alignment with the closest homolog, the uncharacterized tetrameric type AB thioesterase from Shewanella oneidensis, the important catalytic residues of YigI were predicted as following: Asp69, Gln56 and His60. Enzyme activity decreased after the site-direct mutagenesis of those residues. Cytoplasmic acetyl-coenzyme A hydrolase (CACH), also known as acyl-CoA thioesterase12 (Acot12), plays important cellular roles in mammalian fatty acid metabolism through the hydrolysis of acyl-CoA thioesters. Unlike YigI, besides two hotdog domains, CACH contains a steroidogenic acute regulatory (START) domain. The START domain is known as a lipid transporter. Thus, the function and regulation of CACH is f special interest. Chapter three of this thesis focuses on the function of the tandem hotdog-fold domains of CACH. The double hotdog-fold unit of CACH was expressed in E. coli and purified. Surprisingly, it was shown to display highest activity towards isobutyryl-CoA rather than acetyl-CoA.

The Haloacid Dehalogenase Superfamily (HADSF) is a ubiquitous family of enzyme with more than 32,000 members. A variety of reactions are catalyzed by HAD members, but a majority of HAD members are phosphatases. Dephosphorlyation of organophosphate metabolites are carried out in the conserved Rossmann-like core domain. The Rossman-like core domain houses the conserved active site residues necessary for catalysis and also supports domain inserts associated with substrate binding. This dissertation will focus primarily on investigating fold fitness and domain dynamics of HAD proteins more specifically three C2 capped proteins. The first part will focus on whether or not the prevailing tenet that Catalytic Function Comes at the Expense of Protein Fold Stability is applicable to the HADSF phosphatase. To test function at the cost of stability site directed mutagenesis was used to replace catalytic residues and used an array of techniques (CD/Fluorescence/SAXS) to monitored denaturation to evaluate stability in the resulting mutants. From denature results we conclude that although most catalytic residues play no role in stability, good or bad, one in particular Asp2 of DXD squiggle shows to be necessary for not only activity but stability as well. To identify what about the residue is essential to stability the side chain and interacting residues were examined. The Rossmann-like catalytic core domain is a robust domain, and to test the co-evolution of the cap and catalytic domains as a coupled folding and catalytic units will be examined. The structural co-dependency was tested by creating truncations of the catalytic and cap domains and identifying stability and activity of each domain and chimeric C2 capped proteins. Thermal denature of truncations and chimeric proteins reveal stability of each domain separately while the chimeric stability establish a robust core Rossmann-like catalytic domain. As previously discussed the cap domain increases function by increasing

This dissertation contains simulations of chemical catalysis in both biological and heterogeneous contexts. A mixture of classical, quantum, and hybrid techniques are applied to explore the energy profiles and compare possible chemical mechanisms both within the context of human and bacterial enzymes, as well as exploring surface reactions on a metal catalyst. A brief summary of each project follows. Project 1 — Bacterial Enzyme SpvC The newly discovered SpvC effector protein from Salmonella typhimurium interferes with the host immune response by dephosphorylating mitogen-activated protein kinases (MAPKs) with a -elimination mechanism. The dynamics of the enzyme substrate complex of the SpvC effector is investigated with a 3.2 ns molecular dynamics simulation, which reveals that the phosphorylated peptide substrate is tightly held in the active site by a hydrogen bond network and the lysine general base is positioned for the abstraction of the alpha hydrogen. The catalysis is further modeled with density functional theory (DFT) in a truncated active-site model at the B3LYP/6-31 G(d,p) level of theory. The truncated model suggested the reaction proceeds via a single transition state. After including the enzyme environment in ab initio QM/MM studies, it was found to proceed via an E1cB-like pathway, in which the carbanion intermediate is stabilized by an enzyme oxyanion hole provided by Lys104 and Tyr158 of SpvC. Project 2 — Human Enzyme CDK2 Phosphorylation reactions catalyzed by kinases and phosphatases play an indispensable role in cellular signaling, and their malfunctioning is implicated in many diseases. Ab initio quantum mechanical/molecular mechanical studies are reported for the phosphoryl transfer reaction catalyzed by a cyclin-dependent kinase, CDK2. Our results suggest that an active-site Asp residue, rather than ATP as previously proposed, serves as the general base to activate the Ser nucleophile. The corresponding transition state features a

The haloacid dehalogenase superfamily (HADSF) is one of the largest known enzyme superfamilies, having more than 32,000 nonredundant members. Enzymes in this superfamily catalyze a number of different chemical reactions including dehalogenation, phosphoryl transfer, and hydrolysis of phosphate esters and phosphates. The vast majority of HADSF members are phosphatases, which contain a highly conserved core domain that is constructed to catalyze hydrolysis reactions of phosphorylated sugars or nucleotides by using an active site Asp nucleophile and a Mg2+ cofactor. Most phosphatases possess a cap domain, which in association with the core domain assists active site desolvation and binds the region of the substrate that is displaced by water upon Asp nucleophilic attack at the phosphoryl group. In the studies described in this Dissertation, two nucleotidases (AphA and mdN) were chosen to examine how the core Rossmann fold of HADSF members stabilizes the transition state of phosphoryl transfer IV reactions and conserves the stable catalytic scaffold under the pressure of evolutionary changes that expand substrate range. Another phosphatase, YidA, was subjected to studies in order to gain information about how accessory cap domains serve as sophisticated substrate recognition sites. AphA is a nonspecific acid phosphohydrolase (NSAP) from Escherichia coli., which is secreted in the periplasmic space. Although AphA has the highest activity towards hydrolysis of nucleotide phosphates (kcat/Km ~ 105 M-1s-1), it does not yet have an identified biological function. The crystal structures of the apo enzyme as well as complexes of AphA with products of nucleotide monophosphate hydrolysis and with substrate/intermediate analogs have been elucidated. Earlier analysis of these crystal structures shows that a specific hydrophobic substrate binding pocket, formed by Phe56 and Tyr193, exists in AphA. The results of solvent isotope studies carried out in the research effort described i

The HaloacidDehalogenase Enzyme Superfamily (HADSF) is a ubiquitous family of enzymes. Presently, more than 45,000 deposited gene sequences encode proteins of the HADSF, and only a fraction of these have defined structure and/or function. The work described in this thesis focuses on function determination in several members of the HADSF. An integrated bioinformatic-protein structure-enzyme mechanism approach was used to differentiate and track D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GHMB) and histidinol phosphatephosphatase (HisB)orthologues; 2-keto-3-deoxynononic acid 9-phosphatephosphatase (KDN9PP) and 2-keto-3-deoxy-D-manno-octulosonic acid 8-phosphatephosphatase (KDO8PP)orthologues; inorganic pyrophosphatase and Β-phosphoglucomutase(Β-PGM) orthologues. In addition, a structure-function/bioinformatic analysis was carried-out on the bifunctional 1,3-diposphoglycerate acyltransferase/Cys-S-glyceryl-3-phosphate phosphatase (FKBH). Each study began with the examination of the genome context of the encoding gene of the target HADSF member. Based on this analysis possible catalytic functions were posited. In vitro activity assays were then applied to test possible substrates. Having identified a potential physiological substrate the X-ray structure of the enzyme-substrate (or substrate analog) complex was determined. From this structure the substrate recognition residues were identified. These residues were replaced by site directed mutagenesis and the impact on substrate binding and catalysis was determined by measuring the steady-state kinetic constants kcatand kcat/Km for each of the mutant enzymes. Residues shown to be important were used as sequence markers to identify among the sequence homologues identified in BLAST searches the most confidently defined orthologues. The final step used in the function annotation procedure was to examine the genome context of the encoding gene of each putative orthologue. These data were then used to for

My doctoral research began with the function assignment to two domains of fusion protein BT4699, from Bacteriodes thetaiotaomicron, BF1314 from Bacteriodes fragilis and PG1653 from Porphyromonas gingivalis. BT4699, however, is insoluble, so its orthologue BF1314 and PG1653 were prepared and studied. The fusion proteins consist of a domain that belong to a large family of phosphatases known as Haloacid Dehalogenase (HAD) superfamily, and a domain belong to the hotdog-fold thioesterase superfamily. The genes encoding the fusion proteins located at an operon that encoding enzymes involved in the menaquinone biosynthetic pathway. So we assumed that the thioesterase domain is connected to the biosynthesis of menaquinone, which sever as a crucial role in the anaerobic bacteria respiratory chain. While the HAD domain is involved in hydrolysis of pyrophosphate produced by MenA in the lower segment of the pathway. The thioesterase domain was predicted to catalyze the hydrolysis of 1,4-dihydroxyl-2-napthoyl-CoA (DHNA-CoA) to 1,4-dihydroxyl-2-napthoate (DHNA) as the precursor of menaquinone synthesis. To study the function of thioesterase domain, the putative substrate 1,4-dihydroxy naphthoyl-CoA was prepared by chemienzymatic strategy. The structure was confirmed by H NMR and mass spectrum. HPLC analysis indicated that DHNP-CoA is very unstable, which could be self-hydrolyzed within 16 hrs at pH=7.5. By using a combination of techniques like steady-state kinetics, HPLC, and NMR, the function of thioesterase domain of the fusion protein were investigated. Steady-state kinetics studies indicated that PG1653 has high activity over DHNP-CoA. HPLC and H-NMR proved the formation of hydrolyzed products. The high specificity of BF1314 and PG1653 over DHNP-CoA testified the function of thioesterase domain. The function of HAD domain is not that as we assumed. It didnt even show any activity towards pyrophosphate. PG1653 fusion protein did not have any phosphatase activity due to the m

My doctoral research primarily focuses on the Escherichia coli hotdog-fold thioesterases ydiI and ybgC. The ydiI gene is colocalized in the operon ydiHIJ. A substrate screen provided evidence that YdiI prefers aryl-CoA substrates and discriminated against the analogous aryl-ACP, unlike the paralog YbdB which works on 2,4-DHB-EntB in the enterobactin synthesis pathway. A bioinformatic approach showed that in some bacteria, ydiI is colocalized with genes from the menaquinone pathway. YdiI was shown to catalyze the hydrolysis of DHNA-CoA with physiological relevance (kcat/KM ~ 105). Furthermore, the E. coli YdiI- strain was shown to have perturbed growth, in good agreement with other menaquinone enzyme knockout experiments. Taking into account the evidence provided, YdiI is likely the DHNA-CoA thioesterase in the menaquinone pathway. Within the substrate binding pocket, changes to the YdiI Val68 to the YbdB equivalent of Met resulted in perturbed catalytic efficiency towards lauroyl-CoA, however not towards its physiological substrate DHNA-CoA. The YbdB M68V mutant resulted in increased efficiency towards lauroyl-CoA and DHNA-CoA, seemingly making it a gatekeeper. Secondly, YdiI catalyzes the hydrolysis of acyl-CoA's using a Glu63, His54, and Gln48 triad determined by mutagenesis experiments. The utilization of 18-O incorporation and rapid-quench techniques provided insight into YdiI's catalytic mechanism. A single phase multiple turnover reaction and the incorporation of 18-O in single turnover reactions suggests that ydiI uses a general base catalytic mechanism where Glu63 is the activating residue. The second E. coli hotdog-fold thioesterase protein discussed within is YbgC. YbgC is encoded by the first ORF of the tol-pal gene cluster. Previous work on the H. influenzae homologue demonstrated affinity for short chain acyl-CoA's and tandem affintity purificiation experiments showed E. coli YbgC co-purifies with ACP. No previous attempts to screen for acyl-ACP have be

The lignin peroxidase promoted α-carbon β-carbon bond cleavage of two diastereomeric pairs of dimeric lignin model compounds were investigated to determine which structural units are more readily cleaved. These model compounds, β-1 (1,2-diaryl-1,3-propanediol) and β-O-4 (1-diaryl-2-aryloxy-1,3-propanediol), represent the most common structural units present in the plant cell wall polymer lignin. The lignin peroxidase catalyzed reaction mechanism was shown to parallel two mechanistically well understood systems, the cerric (IV) ammonium nitrate promoted chemical reacton, and the dicyannoanthracene promoted photochemical reaction, both of which proceed by a SET mechanism. Product profiles and kinetic rate constants were determined and compared for all four model compounds by HPLC and Stopped Flow kinetic techniques.

Hotdog-fold acyl-CoA thioesterases (ACOTs) are found in all three kingdoms of life (Archaea, Bacteria and Eukarya). All the hotdog-fold thioesterases share common catalytic scaffold and function: hydrolysis of acyl-CoA or acyl-ACP (acyl carrier protein). However the biological roles that they perform vary greatly, different substrate specificity and catalytic efficiency assign unique set of biological functions to each of them, which is further defined by tissue distribution, cellular location, protein partners and regulators. The focuses of my doctoral studies, described in this dissertation, are two members of the human hotdog-fold thioesterase superfamily: human THEM4 and human THEM5. The genes encoding hTHEM4 and hTHEM5 are two adjacent brothers in the chromosome 1q21.3. Expression of truncated hTHEM4(Δ39) in E. coli produced a homogenous product. In vitro activity analysis proved that hTHEM4 is a high activity, broad substrate range, acyl-CoA thioesterase. In addition to hydrolysis of short and long chain acyl-CoAs (kcat/KM value: 104 to 106 M-1s-1), it also catalyzes the hydrolysis of myristoyl-ACP and palmitoyl-ACP. The X-ray structure of hTHEM4(Δ39) bound with an inert fatty acyl-CoA analog revealed that N-terminal domain is two separate helix inserts coupled with a disordered extended loop. In order to understand the relationship between hTHEM4 and Akt1, the effect of purified (fl)hTHEM4 and hTHEM4(Δ39) on the phosphorylation (activation) and activity of full-length and truncated Akt1 were tested at GlaxoSmithKline. The results showed that (fl) hTHEM4 has a small but significant direct effect on Akt1 catalytic activity, and also on Akt1 activation at both Thr308 (by PDK1) and Ser473 (by mTORC2). The co-immunoprecipitation experiment was also carried out to investigate the physical association between hTHEM4 and Akt1. Taken together, hTHEM4 and Akt1 are associated with each other, and the effect (inhibition or activation) is concentration-depe

My doctoral research primarily focuses on two hotdog-fold thioesterases, EntH (also known as YbdB) from E. coli, and BFIT2 from Homo sapiens. The EntH (YbdB) gene is included in a large gene cluster that encodes the enzymes of the biosynthetic pathway leading to enterobactin. Building on the hypothesis that EntH might function in a house-keeping' role by liberating misacylated EntB, two potential pathways to EntB misacylation were identified, one involving the phosphopantetheinyl transferase EntD and the other involving 2,3-DHB-AMP ligase EntE. EntH displays thioesterase activity towards a variety of acyl and aryl-holo EntB adducts. Lastly, It was shown that EntF acts on the 2,3-DHB-holo-EntB quickly, but not quickly on misacylated EntB adducts.tandem hotdog-fold thioesterase domains and a C-terminal steroidogenic acute regulatory protein related lipid transfer (START) domain. The expression of BFIT2 is induced during the thermogenesis transition of brown fat tissue. The expression of the recombinant BFIT2 in transfected HEK cells was confirmed by Western blot analysis. The recombinant BFIT2 contains a N-terminal His6-tag and epitope, which was found to be susceptible to posttranslational removal. The recombinant N-terminal (minus residues 1-34) truncated mutant was found not to undergo posttranslational cleavage, thus suggesting that the N-terminal region is a signal sequence. A chimeric protein BFIT2 N(1-42)-GFP was shown by confocal microscopy to co-locate with the mitochondria. The BFTI2 precursor was shown to be taken up by freshly isolated HEK cell mitochondria and cleaved to the mature form. These results confirmed that the N-terminal region of BFIT2 functions as MTS. During the thermogenesis transition of brown fat tissue, BFIT2 might function to restore the balance between free CoA and fatty acyl-CoA by hydrolyzing the long to medium chain fatty acyl-CoAs. Consistent with this hypothesis, BFIT2 was found to be much more active towards palmitoyl-CoA, myristo

The investigations carried out in my doctoral studies focus on (1) applications of pyridinium salt photochemical and tandem ene-yne-ene metathesis reactions to the synthesis of the natural products (+)-lactacystin and lepadiformine C, and (2) the preparation of a novel inhibitor for the class II Giardia lamblia fructose-1,6-bisphosphate aldolase. New developments in the area of pyridinium salt photochemistry were explored in the context of approaches to the natural product (+)-lactacystin. The results of this effort show that irradiation of a substituted 1,2-cyclopenta-fused pyridinium salt in aqueous solution followed by treatment with sodium bicarbonate leads to selective production of an unusual, structural and functional complex, and stereodefined tetracyclic carbamate. This substance contains structural features and a functionality array that would make it applicable as a late stage intermediate in a synthesis of the proteosome inhibitor, lactacystin. A novel strategy was developed for the synthesis of lepadiformine C, a structurally interesting, biologically active marine alkaloid derived from the marine organism Claveline moluccensis. that has a tricyclic structure. The approach, which begins with l-proline, relies on implementation of a ruthenium-alkylidene catalyzed, tandem ene-yne-ene metathesis process to construct the tricyclic structure. Class I and class II fructose-1,6-bisphosphate aldolases (FBPA), enzymes that exhibit no amino acid sequence homology and utilize different catalytic mechanisms, promote the retro-aldol conversion of fructose-1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate as part of energy producing glycolysis pathways in bacteria, protists and humans. The mammalian class I FBPA employs a Schiff base mechanism, involving an active site lysine amine group, whereas the parasitic protozoan Giardia lamblia relies on a class II FBPA that utilizes an active site Zn+2 to stabilize the forming enolate of d