The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps: the hydrolysis of hydantoin to N-carbamylamino acid by an hydantoinase or dihydropyrimidinase enzyme, followed by conversion of the Ncarbamylamino acid to the corresponding amino acid by an N-carbamoylase enzyme. This biocatalytic process has been successfully applied in several industrial processes for the production of enantiomerically pure amino acids used in the synthesis of pharmaceuticals, insecticides, hormones, and food additives. P. putida RU-KM3S was selected for study based on inherent high levels of hydantoinase and N-carbamoylase activity. Subsequent biocatalytic analysis of the enzyme activity within this strain revealed unique properties thus prompting further characterization. The main focus of this research was the isolation of the genes encoding the hydantoin-hydrolysing pathway in RU-KM3S. A genomic library was constructed and screened for heterologous expression of the hydantoin-hydrolysing enzymes. However, this approach was unsuccessful prompting the use of transposon mutagenesis in order to circumvent the drawbacks associated with complementation studies. The enzymes responsible for hydantoin-hydrolysis were identified by insertional inactivation as a dihydropyrimidinase and b-ureidopropionase encoded by dhp and bup respectively. A third open reading frame, encoding a putative transport protein, was identified between the dhp and bup genes and appeared to share a promoter with bup. Analysis of the amino acid sequence deduced from bup and dhp substantiated the distinctive properties and potential industrial application of the L-enantioselective b-ureidopropionase and provided targets for potential optimisation of the substrate-selectivity and activity of the dihydropyrimidinase by site directed mutagenesis. Several transposon-generated mutants with an altered phenotype for growth on minimal medium with hydantoin as the sole source of nitrogen were also isolated. Analysis of the insertion events in these mutants revealed disruptions of genes encoding key elements of the Ntr global regulatory pathway. However, inactivation of these genes had no effect on the dihydropyrimidinase and b-ureidopropionase activity levels. An additional mutant in which the gene coding for the dihydrolipoamide succinyltransferase, which is involved in the TCA cycle, was isolated with reduced levels of both dihydropyrimidinase and b-ureidopropionase activities. These results indicated that the hydantoin-hydrolysis pathway in RU-KM3S is regulated by carbon rather than nitrogen catabolite repression. This was confirmed by the reduction of hydantoin-hydrolysis in cells grown in excess carbon as opposed to nitrogen. Identification of a putative CRP-binding site within the promoter region of these enzymes further supported the regulatory role of carbon catabolite repression (CCR). As CCR in Pseudomonads is poorly understood, elucidation of the mechanism by which the hydantoinhydrolysing pathway in RU-KM3S is regulated would provide valuable insight into this complex process.

Cytochrome P450 (GYP) are a family of membrane-bound enzymes which form a component of the mixed function oxidase (MFO) system and are involved in the metabolism of many endogenous and exogenous compounds and, as such, play key roles in many physiological, pharmacological and toxicological processes. Due to the diversity of their substrates, it is essential to gain an understanding of how their activities can be modulated. The membrane lipid environment of CYP isoforms has proved to be an essential component for their optimal activities and consequently investigations into this aspect of modulation are fundamental to many areas of research. The work described in this thesis investigates 3 methods for the modulation of hepatic microsomal membrane lipids in order to relate 3 components of the lipid bilayer to the associated GYP isoform activities. The effect of incubating post-mitochondrial fractions with a range of phospholipase A2 (PLA2) concentrations at 37 °G on 4 CYP-catalysed reactions during microsomal fraction preparation was examined. The size of the membrane free fatty acid (FFA) pool was found to increase substantially following PLA2 treatment, and the main hydrolysis product was revealed to be arachidonate. Although the bulk fluidity of the membrane was unchanged at specific PLA2 concentrations, differential sensitivities of the GYP isoforms were observed at these concentrations. By assaying the activity of the NADPH-P450 reductase component of the MFO independently, in addition to using an oxygen surrogate to by-pass the reductase enzyme, it was concluded that it was specifically the GYP proteins that were susceptible to the membrane modulation which consequently lead to the observed decreases in the rates of reaction. The subsequent removal of the FFA pool with bovine serum albumin (BSA) was found not to restore the GYP isoform activities. It has been suggested that the inhibitory effect of FFAs may be due to conformational changes in the lipid environment of the isozymes. Also, hydrolysis of membrane phospholipids by PLA2 activity results in the formation of FFAs and Iysophospholipids, both of which can affect substrate partitioning within a membrane. Thus, the remaining lysophospholipids following BSA treatment may have contributed to the decreased activities of the GYP isoforms. A range of PLA2 concentrations at the lower incubation temperatures of 10 °G and 20 °C were used to retailor microsomal lipids in order to investigate the possibility that the GYP activities were associated with specific lipid domains. As with the 37°G incubation, the main hydrolysis product within the FFA pool was found to be arachidonate. Although arachidonate release was not significantly affected at any of the incubation temperatures, the activities of individual GYP isoforms were significantly decreased with increases in incubation temperature at specific PLA2 concentrations. Kinetic studies suggested that PLA 2 treatment of the microsomal membrane modulated CYP activity through the hydrolysis of associated lipid domains, and not by competitive inhibition by the endogenously released arachidonate. In an attempt to elucidate it microsomal membrane fatty acid saturation affected the activities of associated CYP isoforms, a preliminary study was conducted in which membrane lipid species were chemically modified by the use of catalytic hydrogenation. Membrane fractions were successfully hydrogenated, particularly long-chain fatty acids such asarachidonate. The activities of those CYP isoforms investigated were modulated to varying degrees upon hydrogenation of the membrane environment. It was concluded that the use of such catalysts should provide a suitable system for investigations fatty acid unsaturation and CYP activities. Finally, as phosphatidylcholine (PC) has been shown to be important for certain CYP isoform activities, a preliminary study was carried out to determine the effect of the biosynthetic pathway for PC synthesis on mouse hepatic MFO activity. Using microsomal fractions obtained from a transgenic strain in which one of the pathways for PC synthesis was absent, it was found that the PC fatty acid profile was changed compared to that of the wildype fractions. Additionally, the activities of certain CYP isoforms were affected by the biosynthetic origin of PC synthesis. This sensitivity of specific isozymes to the PC biosynthetic pathway may have also been sex-dependent. The work described in this thesis suggests several strategies which could be exploited further to refine and improve experimental studies of CYP isoforms. The results obtained support the concept that the membrane composition and environment of CYP isoforms differentially affects their activities. Due to the complexity of these remarkable biochemical systems, further studies are clearly required to define the membrane properties involved in the modulation of CYP activity.

CYP3A4 is the most abundant P450 enzyme expressed in the human liver and it is responsible for the metabolism of approximately 50% of all clinically administrated drugs. The CYP3A4 gene is transcriptionally regulated by xenobiotics and previous work has demonstrated the first 300bp of proximal promoter to be the minimal requirement for such activation. Several nuclear receptors (CAR, SXR) have been shown to be involved in the induction of the CYP3A4 gene. The aim of this work was to further delineate the molecular basis of CYP3A4 gene expression. In vitro DNase I footprinting was carried out using HepG2 nuclear extracts to map the sites of DNA-protein interaction within the -301/+7 region of the CYP3A4 gene. Putative protein assignment for these sites using in silico analysis revealed the potential binding of transcription factors previously shown to be involved in the regulation of other CYP genes (Sp1, HNF3 and C/EBP?) at the identified protein-DNA interaction sites. These regulatory regions were then disrupted by mutagenesis and their functional effect assessed by transient transfections of reporter gene plasmids into HuH7 hepatoma cells. Statistically significant reductions of reporter gene expression were observed when putative C/EBPa and HNF sites were altered, in both the basal and rifampicin (SXR ligand) induced states. This finding suggests the involvement of proteins binding at these sites in the regulation of the CYP3A4 gene expression. An examination of the CYP3A4 promoter (-1056/+7) from 11 human DNA samples exhibiting a 14.3 fold variability in CYP3A mediated metabolism failed to show the presence of mutations. Protein-DNA interaction analysis were carried out within the newly identified CYP3A4*IE and CYP3A4*1F alleles as well as the CYP3A4 *1B allele. The results implicate the Spl transcription factor in the regulation of the CYP3A4 gene, albeit at a more distal binding site. The findings described in this thesis suggest a substantial involvement of transcription factors other than SXR/CAR in expression of CYP3A4. Because of the polymorphic expression of several liver- and hormone-dependent transcription factors their role in CYP3A4 regulation must be taken into account to understand drug-induction mechanisms and assess variability in inter-individual drug response.

Cette recherche a été entreprise dans le "but d'apporter certaines précisions sur les mécanismes d'adaptation des enzymes pancréatiques à la composition du régime décrits par GROSSMAN(52). Dans une deuxième partie, notre travail a été orienté vers l'étude des mécanismes physiologiques qui régissent la sécrétion et la synthèse des enzymes digestifs du pancréas. Ces recherches, menées tant in vivo qu'in vitro, ont englobé l'action de différents stimuli; repas, pancréozymine, gastrine, histamine, sérotonine et acétylcholine.

M.Sc. (Biochemistry) / Please refer to full text to view abstract

Magister Scientiae - MSc / This study represents the first structural investigation of any type III glutamine synthetase (GS). The GS, GlnA, from the medically important opportunistic human pathogen Bacteroides fragilis was studied with a view to better understanding its structure/functioning in relation to the extensively characterised GSIs. GSIIIs are the most recently discovered family of GSs and are the most phylogenitically distant GSs from the GSIs. Images (160) of negatively stained rGlnA, expressed in E.coli YMC11 (glnA-), were recorded at 50K magnification using a Leo 912 operating at 120kV with energy filtering coupled to a 4 megapixel CCD camera. An angular refinement based reconstruction strategy was adopted using SPIDER. A reconstruction based on an ab initio starting model, derived by a common-lines based simultaneous minimization of rotationally invariant K-mean clustered class averages, converged to the same structure as a reconstruction based on a GSI starting model to a resolution of 2.1nm as determined by Fourier shell correlation). In contrast to preliminary EM observations, which identified GlnA as a hexamer, this work has revealed a dodecameric structure, with subunits (82.8KDa) arranged in two opposing hexagonal rings with distinct handedness. This is similar to the quaternary structure of GSIs and GlnTs except that the complex is 50% longer and the two rings are not symmetrically related. They differ not only in diameter (16.5 or 15.0nm) but also the degree of separation of subunits and as such the particle possesses only C6 and not D6 symmetry. The finding that particles lie in a preferred orientation, with the larger ring in contact with the carbon support, accounted for this asymmetry, through partial staining. Hexameric views, with similar overall arrangement but larger size in comparison to GSI, were also observed. However, it was uncertain whether these were true hexamers resulting from dissociation of the dodecamers or were a consequence of partial staining. Homology modelling was also undertaken in an attempt to predict the structure of GlnA based on GSI, with a view to interpreting the low resolution EM structure. Due to the failures of state of the art algorithms in detecting the distant homologies between GS families, manual profile-based alignment strategies, incorporating structural information, were employed. Through the first full length alignments of GS sequences from all four families, conservation of all active site residues, core active site αβ barrel fold motifs, and additional previously unreported regions was demonstrated. Docking of these homology models into the 3D structure confirmed the presence of the αβ barrel fold predicted by the bioinformatic analysis of the sequences alone, thus, identifying the indentations between subunits in the volume as putative active sites. In addition to providing unequivocal proof that GlnA is a GS and confirming the presence of putative αβ barrel active site folds, this work has made steps towards understanding the regulation of this enzyme. It is hypothesised that GlnA occurs as both active hexamer and an inactive dodecamer, the interconversion of which, is thought to represent a means of reversible post-translational regulation. / South Africa

Magister Scientiae - MSc / The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common αββα structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the αββα-αββα fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm. The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 Å. Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost – either by specific proteolysis or autolysis - an active helix is formed. / South Africa

No description available.

No description available.

In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.