Streptococcus pneumoniae is a commensal inhabitant of the human nasopharynx that can sometimes become pathogenic and cause diseases such as pneumonia, otitis media and meningitis. Carbohydrate metabolism is a critical component of S. pneumoniae virulence. Among the myriad of carbohydrate-specific pathways involved in the host-pneumococcus interaction, the N-glycan foraging pathway stands out because of its direct implication in numerous aspects of virulence such as fitness, adhesion/invasion and impairment of the host immune response. Much of the literature has been focussed on the importance of step-wise depolymerisation of N-glycans by the enzymes NanA, BgaA and StrH. However, the importance of the liberation of N-glycans from host glycoconjuguates and their intake by the bacterium has yet to be examined. We have identified a Carbohydrate Processing Locus (CPL) that is highly conserved throughout a large number of Firmicutes and whose individual components appear widespread in bacteria that we hypothesize is active on host N-glycans. This locus encodes for two putative α-mannosidases GH92 and GH38, a characterised α-mannosidase GH125, a putative β-hexosaminidase GH20C, a putative α-fucosidase GH29 and a ROK (Repressor, Open reading frame, Kinase) protein. The genomic context of CPL orthologues suggests that an endo-β-N-acetylglucosaminidase (EndoD) and an ABC transporter (ABCN-glycan) are functionally associated with this locus. Based on our bioinformatic analyses and known functions of these proteins we hypothesize that the CPL encodes a concerted pathway responsible for the liberation, transport, and processing of N-glycans. The objective of this research is to characterize the putative components of this pathway and assess their implication in virulence. Specific focus on ABCN-glycan demonstrated its specificity for a range of N-glycans liberated by EndoD, shedding light on a novel import system for branched N-glycans. Furthermore, we provided evidence that GH92 is an α-1,2-mannosidase that likely removes the terminal mannose residues found on high-mannose N-glycans. EndoD and GH92 are shown to participate in virulence in mice; however, their role in virulence has yet to be determined. This work will significantly advance the construction and validation of a model of N-glycan processing by S. pneumoniae. As the components of this model pathway are conserved amongst a wide variety of bacteria, this work is of fundamental relevance to understanding how microbes from various environments degrade and metabolize N-glycans. / Graduate

The degradation of carbohydrates is essential to many biological processes such as cell wall remodelling, host-pathogen defense, and energy synthesis in the form of ATP. Several of these processes utilize carbohydrate-active enzymes to accomplish these goals. Studying the degradation of polysaccharides by carbohydrate-active enzymes synthesized by microbes has allowed us to further understand biomass conversion. A portion of these polysaccharides consists of β-1,3-linked glucose (i.e. β-1,3-glucan), which is found in plants, fungi, and brown macroalgae. The hydrolysis of β-1,3-glycosidic linkages is catalyzed by β-1,3-glucanases, which are present in six different glycoside hydrolase (GH) families: 16, 17, 55, 64, 81, and 128. These enzymes play important biological roles including carbon utilization, cell wall modeling, and pathogen defense. This study focuses on a gene from Bacillus halodurans encoding for a multi-modular protein (BhLam81) consisting of a glycoside hydrolase from family 81 (BhGH81), a carbohydrate-binding module (CBM) from family 6 (BhCBM6), and a CBM from family 56 (BhCBM56). Previously, thorough structural and substrate-specific characterization has been carried out on BhCBM6. This CBM binds the non-reducing end of β-1,3-glucan. A member of CBM family 56 has been shown to recognize and bind the insoluble β-1,3-glucan, pachyman, however it is structurally uncharacterized. A glycoside hydrolase belonging to family 81 from Saccharomyces cerevisiae has been previously shown to degrade the β-1,3-glucans, laminarin and pachyman, however the structure of this enzyme was not determined. Recently, a member of GH family 81 has been structurally characterized; however, substrate-specificity was not determined in that study. Therefore, this study concentrated on two goals: Determining the substrate-specificity of BhGH81 and BhCBM56, and solving the structure of BhGH81 and BhCBM56 in order to gain insight into the molecular details of how they recognize and act on their substrate(s). The deoxyribonucleic acid (DNA) encoding for these modules were dissected by restriction digest from B. halodurans genomic DNA and recombinantly expressed in Escherichia coli (E. coli) as separate constructs. Both BhGH81 and BhCBM56 were purified and their crystal structures obtained. BhGH81 and BhCBM56 were solved to 2.5 Å resolution by single-wavelength anomalous dispersion (SAD) and to 1.7 Å resolution by multi-wavelength anomalous dispersion (MAD), respectively. In order to determine the substrate-specificity of BhGH81 and BhCBM56 and speculate on the molecular details of how they recognize and act on their substrate(s), substrate-specificity tests were combined with structural analysis for both of these modules. By using qualitative depletion assays, quantitative depletion assays, and affinity electrophoresis, it was revealed that BhCBM56 binds both insoluble and soluble β-1,3-glucan. The crystal structure of BhCBM56 revealed that it is a β-sandwich composed of two antiparallel β-sheets consisting of five β-strands each. By comparing BhCBM56 to a β-1,3-glucan binding protein from Plodia interpunctella (βGRP) a putative substrate-binding cleft on the concave side of the β-sandwich created by a platform of hydrophobic residues surrounded by several polar and charged residues was revealed. This comparison also allowed for speculation of the amino acids (W1015, H965, and D963) that are potentially essential for recognition of β-1,3-glucan substrates by BhCBM56. Activity of BhGH81 on β-1,3-glucans was confirmed by both thin-layer chromatography and product analysis using high performance anion exchange chromatography. The high performance anion exchange chromatography of BhGH81 hydrolysis suggested it has both exo and endo modes of action. The crystal structure of BhGH81 revealed that it consists of domains A, B, and C: A β-sandwich domain (A), a linker domain (B), and an (α/α)6-barrel domain (C). This structure revealed a putative substrate-binding cleft on one side of the (α/α)6-barrel with a blind canyon active site topology. It also revealed two putative catalytic residues, E542 and E546. All GHs from family 81 characterized so far, hydrolyze β-1,3-glucan in an endo acting manner. By comparing the structure of BhGH81 acquired in this study to a cellulase from Thermobifida fusca, which has an endo-processive mode of action, we can speculate that BhGH81 also has an endo-processive mode of action. The structural and biochemical analysis of BhGH81 and BhCBM56 in this study has aided in further understanding the molecular details both GH family 81 and CBM family 56 proteins, as well as the degradation of β-1,3-glucan by multimodular enzymes. Understanding these molecular details could be important for industrial applications such as, engineering a microbial platform for more efficient biofuel production. / Graduate

Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated. / Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.

Background: Relatively little is known about the genomic basis and evolution of wood- feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Les MICI (maladies inflammatoires chroniques de l'intestin) invalident 200 000 personnes en France. La région Nord-Pas-de-Calais est particulièrement touchée par ces affections et les traitements disponibles pour ces pathologies demeurent coûteux et peu nombreux. Des études récentes ont suggéré que le système endocannabinoïde, exprimé au seing du tractus gastro-intestinal, est une cible thérapeutique prometteuse pour le traitement des MICI. Ce système se compose des récepteurs aux cannabinoïdes CB1 et CB2, des ligands endogènes de ces récepteurs, notamment l'anandamide et le 2-arachidonoylglycérol et des protéines impliquées dans l'anabolisme et le catabolisme des ligands. L'anandamide a présenté des capacités à prévenir la colite induite par le TNBS à des rongeurs. Toutefois, in vivo ce composé possède un temps de demi-vie court et est rapidement dégradé par une amidase à sérine, la FAAH (Fatty Acid Amide Hydrolase). Nous avons ainsi envisagé la conception, la synthèse et l'évaluation pharmacologique de nouveau inhibiteurs de la FAAH. L'une de nos molécules, le composé 95, présentant une CI50 sur l'enzyme de 88 nM a ensuite été injectée par voie intrapéritonéale à des souris dont la colite a été induite trois jours plus tard par l'injection intrarectale de TNBS. L'évaluation des scores macroscopiques et microscopiques des dommages causés sur le côlon par l'agent irritant a ensuite été effectuée. L'inflammation du côlon a été significativement réduite chez le groupe de souris ayant été traité par le composé 95, montrant que l'inhibition de la FAAH est une stratégie thérapeutique efficace dans le traitement des MICI. / IBD (Inflammatory Bowel Diseases) invalidate 200 000 persons in France. Nord-Pas-de-Calais region is particularly touched by these diseases and the available treatments for these pathologies are few and expensive. Recent studies have suggested that endocannabinoid system expressed in the gastrointestinal tract, was a promising therapeutic target for the IBD treatment. This system is made up of cannabinoids receptors CB1 and CB2, endogenous ligands of these receptors, notably anandamide and 2-arachidonoylglycerol, and proteins involved in ligands metabolism. Anandamide has shown properties to prevent TNBS-induced colitis in mice. However, in vivo, anandamide possesses a short life time and is rapidly hydrolyzed by a serine amidase, the FAAH (Fatty Acid Amide Hydrolase). In this context, we have considered the design, the synthesis and the pharmacological evaluation of new FAAH inhibitors. One of our molecules, compound 95, inhibiting the enzyme with an IC50 value of 88 nM has been injected intraperitonally to mice which the colitis was induced three days later by intrarectal TNBS-administration. The assessment of macroscopic and microscopic scores of colonic damages was undergone. Colonic inflammation was significatively reduced in the group of mice which has been treated by 95, showing evidence that FAAH inhibition was an effective therapeutic target for the treatment of IBD.

The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.

Cancer cell esterases are often overexpressed and some have chiral specificities different from those of corresponding normal cells. Carboxylesterases in particular are known to be overexpressed in several cancers. Additionally, cancer cells often exhibit high levels of intrinsic oxidative stress that is required for survival and an aggressive phenotype. We hypothesized that these 2 characteristics of cancer cells could be exploited to aid in the detection and treatment of prostate cancer. We have developed a fluorogenic ester probe that is activated by carboxylesterase to help distinguish tumorigenic cells from nontumorigenic prostate cells. Ester prodrugs have the same activation mechanism and have been thought to be a promising approach in cancer therapy. Prodrugs are inactive drugs that can be selectively activated by a specific enzyme. We have developed a chiral ester prodrug strategy using native polyacrylamide gel electrophoresis (n-PAGE) and proteomic methods to compare and identify the esterase profiles of several tumorigenic and nontumorigenic prostate cell lines. Our results showed that cell lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines exhibit differential esterase activity compared with non-tumorigenic RWPE-1 prostate cell lysates when incubated with α- naphthyl acetate or α-naphthyl N-acetyl-alaninate ester substrates and a diazonium salt. We have identified oxidized protein hydrolase (OPH), a serine esterase/protease that catalyzes the removal of N-acylated residues from proteins, to be differentially expressed between some tumorigenic and nontumorigenic prostate cell lines. OPH was found to have high hydrolytic activity towards the S-isomer of α-naphthyl N-acetylalaninate (S-ANAA) chiral ester. LNCaP lysates incubated with N-acetyl-alanyl-p-nitroanilide, a known OPH substrate, had twofold higher OPH activity compared with RWPE-1 lysates. We have also developed and tested novel glutathione depleting prodrugs modeled after S-ANAA that increase oxidative stress and induced apoptosis in tumorigenic prostate cells with little effect on nontumorigenic RWPE-1 cells. These results suggest that ester molecular beacon probes and ester prodrugs may be effective in identifying and treating prostate cancer tumors that overexpress esterases with little effect on normal prostate cells.

N-Acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the “endocannabinoid” signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

Fat facets in Mouse (FAM) or mUSP9x is a deubiquitylating enzyme of the USP class. Knockdown of FAM protein levels in mouse pre-implantation embryos by antisense oligonucleotides is known to prevent embryos from progressing to the blastocyst stage indicating an important role for FAM in early mammalian development. In mammals, the Fam gene is located on the X-chromosome. In mice, the Y homologue, Dffry or usp9y, is expressed exclusively in the testes and maps to the Sxrb deletion (Brown et al., 1998). Sxrb is associated with an early post-natal blockage of spermatogonial proliferation and differentiation leading to absence of germ cells (Bishop et al., 1988; Mardon et al., 1989). The human Y homologue of Fam is closely associated with oligozoospermia (Sargent et al., 1999; Sun et al., 1999) and the human X homologue has been linked to the failure of oocytes to pass through the first meitoc prophase in Turner syndrome (Cockwell et al., 1991; Speed, 1986) Despite these associations, the substrates and precise role of Fam and its homologues in these processes have not yet been defined. Due to the complex nature of Fam expression and the lack of data tying FAM to specific cellular functions, much attention has been paid in identifying interacting partners and cellular targets of FAM activity to aid in the definition of its role in the cell and development. Three common molecular biology techniques were applied here in an attempt to further characterise known interactions of FAM, including interactions with the cell adhesion molecule β-catenin and the protein trafficking pathway proteins epsin-1 and itch. The aim of these investigations was to generate FAM mutants that could abolish individual interactions, enabling investigation of individual interactions in cellular function and development. These experiments failed to identify the amino acids of FAM that were critical for its interactions with β-catenin, epsin-1, or itch. Experiments aimed at characterising a novel ubiquitin-like domain located in the N-terminal half of the FAM protein, did however identify novel interactions of FAM with the three Golgi associated adaptor proteins GGA1, GGA2, and GGA3. Further investigations prompted by this interaction, examined the role of FAM in the trafficking of proteins from the Golgi apparatus. Cellular FAM protein levels were altered either by exogenous expression of FAM protein or knockdown of endogenous FAM using FAM specific shRNA triggers. The cellular protein levels and extent of post-translational modification of eleven lysosomal proteins were monitored in each case. It was found that increased FAM protein levels resulted in decreased cellular protein levels of five of the eleven lysosomal proteins studied. In contrast, a reduction in FAM protein levels was found to result in an increase in the cellular protein levels of eight of the eleven lysosomal proteins. This study provides the first evidence of a deubiquitylating enzyme that is able to interact with the GGA proteins. It is also the first to describe a deubiquitylating enzyme that can affect the biosynthesis of lysosomal proteins and provides valuable new insight into the cellular function of FAM/USP9X. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2008

Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.