MshA is a glycosyltransferase that synthesizes the precursor of mycothiol, a low-molecular-weight thiol found exclusively in Actinomycetes, including the virulent pathogen Mycobacterium tuberculosis (Mtb). The structure of MshA from Mtb (herein coined as TbMshA) and its complex with uridine diphosphate N-acetyl-glucosamine (UDP-GlcNAc) have been solved to resolutions of 2.32 A and 2.89 A respectively. Both structures form two monomers in the asymmetric unit cell and exhibit typical beta/alpha/beta Rossmann folds. Upon binding of UDP-GlcNAc, the C-terminal domain of TbMshA undergoes conformational changes in order to interact with UDP-GlcNAc at the binding site. In addition, ligand-bound TbMshA structure enables the identification of critical residues for enzymatic interactions, especially the residue Glu-353 (E353) at the active site that is believed to serve as a nucleophile in the sugar transfer of TbMshA. In order to verify this, a mutant of TbMshA with a single amino acid mutation from glutamate to glutamine at residue 353 is generated. The mutant (E353Q) has shown reduced enzyme activity by more than four-fold compared to the wild-type TbMshA (Vmax for wild-type is 0.17 plus/minus 0.02 microM sec^-1, whereas Vmax for E353Q is 0.04 plus/minus 0.01 microM sec-1). The kcat/Km for wild-type TbMshA (3.5 plus/minus 1.1 * 10^3 M^-1 sec^-1) is an order of magnitude higher than that of the mutant (0.3 plus/minus 0.1 * 10^3 M^-1 sec^-1), indicating the catalytic efficiency is greatly suppressed by the mutation. Mass spectrometry data also reveals that E353Q is unable to form the product of the reaction catalyzed by the wild-type TbMshA. These findings suggest the important role of Glu-353 in the structure and activity of TbMshA.

The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (??-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. Given the clinical importance of this intracellular thiol, further investigation into the behaviour of ergothioneine appeared warranted. A high level theoretical study was performed to determine the thermodynamic driving force behind the instability of the ergothioneine disulfide, as well as the thermodynamics of the reactions of ergothioneine with a selection of biologically relevant reactive oxygen species. These results were compared to those determined for a glutathione model compound, as well as the related ovothiols. The latter are believed to act as hydrogen peroxide scavengers in vivo and are currently under review as possible therapeutics against oxidative damage. The structural differences between the ovothiols and ergothioneine dramatically affect their reactivity and this study investigates the thermodynamic driving forces behind these differences. Mycothiol is the major thiol found in the Actinomycetales bacteria, which include the causative agent of tuberculosis, and the enzymes which use mycothiol have been identified as important targets for the development of novel antimicrobials. To better understand the in vivo behaviour of mycothiol, a thorough conformational search was performed to determine what, if any, trends exist among the low energy conformers expected to be present in solution. Knowledge of the conformations preferred by mycothiol may aid in the design of substrate-based inhibitors targeted at mycothiol-dependent enzymes. In addition, the efforts towards the identification of a mycothiol-dependent glyoxalase system are described. The glyoxalase system is essential for the detoxification of methylglyoxal, a toxic by-product of glycolysis, and this system would serve as a target for the design of new therapeutics against tuberculosis and other pathogenic Actinomycetales bacteria. In addition to the study of intracellular thiols, this work details a preliminary theoretical study of the thermodynamics of the phosphorylation of proteinaceous serine residues by inositol pyrophosphates in eukaryotic cell-free extracts. It has been postulated that this observed activity may represent a novel signalling pathway in eukaryotes. This study focused on the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of these reactions. This information should contribute to our understanding of this novel cellular phosphorylation process.

The presence of thiols in living systems is critical for the maintenance of cellular redox homeostasis, the maintenance of protein thiol-disulfide ratios and the protection of cells from reactive oxygen species. In addition to the well studied tripeptide glutathione (γ-Glu-Cys-Gly), a number of compounds have been identified that contribute to these essential cellular roles. Many of these molecules are of great clinical interest due to their essential role in the biochemistry of a number of deadly pathogens, as well as their possible role as therapeutic agents in the treatment of a number of diseases. A series of studies were undertaken using theoretical, chemical and biochemical approaches on a selection of thiols, ergothioneine, the ovothiols and mycothiol, to further our understanding of these necessary biological components. Ergothioneine is present at significant physiological levels in humans and other mammals; however, a definitive role for this thiol has yet to be determined. It has been implicated in radical scavenging in vivo and shows promise as a therapeutic agent against disease states caused by oxidative damage. Given the clinical importance of this intracellular thiol, further investigation into the behaviour of ergothioneine appeared warranted. A high level theoretical study was performed to determine the thermodynamic driving force behind the instability of the ergothioneine disulfide, as well as the thermodynamics of the reactions of ergothioneine with a selection of biologically relevant reactive oxygen species. These results were compared to those determined for a glutathione model compound, as well as the related ovothiols. The latter are believed to act as hydrogen peroxide scavengers in vivo and are currently under review as possible therapeutics against oxidative damage. The structural differences between the ovothiols and ergothioneine dramatically affect their reactivity and this study investigates the thermodynamic driving forces behind these differences. Mycothiol is the major thiol found in the Actinomycetales bacteria, which include the causative agent of tuberculosis, and the enzymes which use mycothiol have been identified as important targets for the development of novel antimicrobials. To better understand the in vivo behaviour of mycothiol, a thorough conformational search was performed to determine what, if any, trends exist among the low energy conformers expected to be present in solution. Knowledge of the conformations preferred by mycothiol may aid in the design of substrate-based inhibitors targeted at mycothiol-dependent enzymes. In addition, the efforts towards the identification of a mycothiol-dependent glyoxalase system are described. The glyoxalase system is essential for the detoxification of methylglyoxal, a toxic by-product of glycolysis, and this system would serve as a target for the design of new therapeutics against tuberculosis and other pathogenic Actinomycetales bacteria. In addition to the study of intracellular thiols, this work details a preliminary theoretical study of the thermodynamics of the phosphorylation of proteinaceous serine residues by inositol pyrophosphates in eukaryotic cell-free extracts. It has been postulated that this observed activity may represent a novel signalling pathway in eukaryotes. This study focused on the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of these reactions. This information should contribute to our understanding of this novel cellular phosphorylation process.

No description available.

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Mycothiol (MSH) is unique to mycobacteria as the major low molecular weight cellular thiol responsible for protection of bacteria against oxidative stress. The design of drugs and inhibitors against enzymes of the mycothiol pathway was based on the premise that mycothiol is unique to mycobacteria, and is thus important for its survival. A total of 80 inhibitors designed against enzymes of the mycothiol pathway were screened for inhibition of growth on in vitro growing M. tuberculosis using the BACTEC 460TM assay. The most active compounds were further tested for inhibitory potential of M. tuberculosis within macrophages. Initial screening in the macrophage system was done using the human-like THP1 cell line and then mouse bone marrow-derived macrophages. In this investigation we established that phenothiazine can be exploited as an inhibitor of enzymes of the mycothiol pathway. Although tunicamycin significantly inhibited the growth of M. tuberculosis both in vitro and ex vivo; it was found to be cytotoxic to host macrophages. To this end we provide proof-of-concept that compounds which can inhibit the expression of mycothiol enzymes have potential as anti-tubercular drugs. The response of M. tuberculosis to stress conditions was assessed via LC-MS in which maximal levels of mycothiol were produced during the early time points of exposure to isoniazid. We used mycothiol-deficient (mshA) M. tuberculosis to investigate the role of mycothiol for survival as well as the resultant phenotype when such mutants are exposed to stress conditions. The mshA deletion mutants in M. tuberculosis were resistant to INH at concentrations which inhibited growth in the wild-type strains. We postulated that katG and inhA, the genes involved in INH metabolism, required mycothiol for their activation. Morphological alterations of M. tuberculosis within macrophages were assessed using electron microscopy approaches. In this way we attempted to follow the fate of M. tuberculosis within the phagosomes, and how mycobacteria is processed in phagosomes in terms of replication, survival and degradation. The establishment of a successful infection by M. tuberculosis depends on the initial encounter with host macrophages, which represent the first line of cellular defense against microbial invasion. At the interface between mycobacteria and macrophages, the complex outermost layer of the mycobacterial cell wall probably plays a role in facilitating host cell entry. Under normal conditions (i.e. ingestion of non pathogenic microorganisms), newly formed phagosomes intermingle contents and membrane with the successive compartments of the endocytic pathway (early endosomes, late endosomes, lysosomes) through a complex series of fusion and fission. As they are processed into phagolysosomes, they undergo gradual modifications by specific addition and removal of membrane constituents. In addition, they become acidified due to the vacuolar proton pump ATPase located in the membrane and acquire toxic constituents, including hydrolases that will ultimately destroy bacteria. / AFRIKAANSE OPSOMMING: Mycothiol (MSH) is uniek aan mycobacteria as die belangrikste lae molekulêre gewig sellulêre thiol verantwoordelik vir die beskerming van bakterieë teen oksidatiewe stres. Die ontwerp van dwelms en inhibeerders teen ensieme van die mycothiol pad is gebaseer op die veronderstelling dat mycothiol uniek is aan mycobacteria, en is dus belangrik vir sy oorlewing. 'n Totaal van 80 inhibeerders ontwerp teen ensieme van die mycothiol pad is gekeur vir die inhibisie van groei op in vitro groeiende M. tuberculosis met behulp van die BACTEC 460TM toets. Die mees aktiewe verbindings is verder getoets vir inhiberende potensiaal van M. tuberculosis binne makrofage. Aanvanklike sifting in die makrofage stelsel is gedoen met behulp van die mens-soos THP1 sel lyn dan makrofage afkomstig van muis beenmurg. In hierdie ondersoek het ons vasgestel dat fenotiasien kan gebruik word as 'n inhibitor van ensieme van die mycothiol pad. Alhoewel tunicamycin aansienlik die groei van M. tuberculosis beide in vitro en ex vivo inhibeer, was dit gevind word sitotoksies is vir makrofage. Om hierdie rede het ons bewys-van-konsep wat verbindings dat die uitdrukking van mycothiol ensieme inhibeer, die potensiaal het as anti-tuberkulose dwelms. Die reaksie van M. tuberkulose op stress is geëvalueer deur LC-MS waarin maksimum vlakke van mycothiol gedurende die vroeë tyd punte van blootstelling aan isoniasied geproduseer is. Ons gebruik mycothiol-deficient (mshA) M. tuberculosis om die rol van mycothiol vir oorlewing sowel as die gevolglike fenotipe te ondersoek wanneer sodanige mutanten blootgestel word aan stres kondisies. Die mshA-weglating mutanten van M. tuberculosis was bestand teen INH konsentrasies wat groei geïnhibeer in die wilde-tipe-stamme. Ons veronderstel dat katG en inhA, die gene wat betrokke is in INH metabolisme, mycothiol vereis vir hulle aktivering. Morfologiese veranderinge van M. tuberculosis binne makrofage is beoordeel met behulp van elektronmikroskopie. In hierdie manier waarop ons probeer om die lot van M. tuberkulose te volg binne die phagosomes, en hoe mycobacteria verwerk word in phagosomes in terme van replikasie, oorlewing en agteruitgang. Die vestiging van „n suksesvolle infeksie deur M. tuberculosis hang af van die aanvanklike ontmoeting met host makrofage, wat die eerste lyn 'n sellulêre verdediging teen mikrobiese inval verteenwoordig. Op die grens tussen mycobacteria en makrofage, speel die komplekse buitenste laag van die mikobakteriese selwand waarskynlik 'n rol in die intog van die gasheersel. Onder normale omstandighede (dws inname van non patogene mikroörganismes), nuutgevormde phagosomes meng inhoud en membraan met die opeenvolgende kompartemente van die endositiese roete (vroeë endosomes, laat endosomes, lisosome) deur 'n komplekse reeks van samesmelting en fisie. Soos hulle verwerk word tot phagolysosomes, ondergaan hulle geleidelike veranderinge deur spesifieke optel en verwydering van membraan komponente. Benewens, raak hulle versuur as gevolg van die vacuolair proton pomp ATPase geleë in die membraan en verkry giftige bestanddele, insluitend hydrolase wat uiteindelik bakterieë vernietig.

<p>This thesis describes the synthesis of structures related to the capsular polysaccharide of <i>Neisseria meningitidis</i> serogroup A and the synthesis of analogues of mycothiol, a compound produced by <i>Mycobacterium</i> <i>tuberculosis</i>. The first part of the thesis describes the synthesis of structural elements present in the native capsular polysaccharide of <i>Neisseria</i> <i>meningitidis</i> serogroup A. In this part, an improved synthesis of 2-azido-2-deoxy-D-mannopyranose is included. The second part of the thesis describes the formation of stable C-phosphonate analogues related to the capsular polysaccharide. The last part outlines the formation of analogues of mycothiol, where the syntheses of a bicyclic analogue and a thioglycosidic analogue are described.</p>

This thesis describes the synthesis of structures related to the capsular polysaccharide of Neisseria meningitidis serogroup A and the synthesis of analogues of mycothiol, a compound produced by Mycobacterium tuberculosis. The first part of the thesis describes the synthesis of structural elements present in the native capsular polysaccharide of Neisseria meningitidis serogroup A. In this part, an improved synthesis of 2-azido-2-deoxy-D-mannopyranose is included. The second part of the thesis describes the formation of stable C-phosphonate analogues related to the capsular polysaccharide. The last part outlines the formation of analogues of mycothiol, where the syntheses of a bicyclic analogue and a thioglycosidic analogue are described.

Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH).  MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification.  Therefore, MSH is considered a potential drug target.  The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins).  In this dissertation, we present detailed functional analysis of MshB.  Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including  metal ion availability and oxidative conditions.  MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate.  Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products.  A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate.  Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB.  Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates.  Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment. / Ph. D.

Specialized microbial metabolites are described as low-molecular-weight bioactive compounds, which are dispensable for the growth, evolution, or reproduction of its producer. This group of substances includes the lincosamides, which are produced mainly by the bacteria of the Streptomyces genera. Apart from other precursors, two low-molecular-weight thiols, ergothioneine and mycothiol, are essential participants of the lincosamide biosynthesis. Mycothiol (MSH) serves in this pathway as a source of sulphur, on the other hand, ergothioneine (ESH) constitutes a conjugate with the aminosugar moiety of lincosamide structure. The conjugate is condensed with an activated amino acid, which is catalyzed by an unusual enzyme to form a core of the lincosamide molecule. The objective of this diploma thesis is to isolate the conjugate of ESH and aminooctose, which serves as a substrate of the LmbD biosynthetic protein. Another aim is to study the links between the thiol metabolism and the biosynthesis of three lincosamides, lincomycin, celesticetin, and intervencin, which are produced by different bacterial strains. Bacterial strains were cultivated under laboratory conditions and methods of liquid chromatography with UV and MS detection were used for the analysis. The parameters of the methods were developed...

Myo-inositol is ubiquitous in nature and is found at the structural core of a diverse range of biologically important derivatives, including phosphatidylinositols, inositol phosphates and mycothiol. The synthesis of myo-inositol derivatives is notoriously difficult due to the need to control both regio- and enantioselectivity. As a result, synthetic routes to derivatives of this type are often lengthy and low yielding. The first biosynthetic step in the production of all myo-inositol metabolites is the isomerisation of D-glucose 6- phosphate to L-myo-inositol 1-phosphate as mediated by L-myo-inositol 1-phosphate synthase (INO1). For the protozoan parasite Trypanosoma brucei, INO1 is essential for survival and its version of the enzyme (TbINO1) has a high turnover. This makes TbINO1 an attractive candidate for the biocatalytic production of L-myo-inositol 1- phosphate, and a potential starting point for drastically shortened syntheses of important myo-inositol derivatives. The production of L-myo-inositol 1-phosphate by TbINO1 has been optimised to achieve complete conversion in reaction conditions that facilitate product isolation. Due to problems with an in-batch process, the TbINO1 enzyme was immobilised and the process was transferred to a flow system. This has allowed for production of significant quantities of L-myo-inositol 1-phosphate with a high level of purity. L-myo-inositol 1- phosphate obtained from the flow system has been used to prepare mycothiol glycosylation acceptor, 1,2,4,5,6-penta-O-acetyl-D-myo-inositol, in a concise synthesis with a greatly improved yield over the literature.