Plant sesquiterpene synthases catalyze the conversion of the linear substrate farnesyl diphosphate, FPP, into a remarkable array of secondary metabolites. These secondary metabolites in turn mediate a number of important interactions between plants and their environment, such as plant-plant, plant-insect and plant-pathogen interactions. Given the relative biological importance of sesquiterpenes and their use in numerous practical applications, the current thesis was directed towards developing a better understanding of the mechanisms employed by sesquiterpene synthases in the biosynthesis of such a diverse class of compounds. Substrate preference for sesquiterpene synthases initially isolated from Nicotiana tabacum (TEAS), Hyoscyamus muticus (HPS) and Artemisia annuna (ADS) were optimized with regards to a divalent metal ion requirement. Surprisingly, careful titration with manganese stimulated bona fide synthase activity with the native 15-carbon substrate farnesyl diphopshate (FPP) as well as with the 10-carbon substrate geranyl diphosphate (GPP). Reaction product analysis suggested that the GPP could be used to investigate early steps in the catalytic cascade of these enzymes. To investigate how structural features of the sesquiterpene synthases translate into enzymatic traits, a series of substrate and active site residue contacts maps were developed and used in a comparative approach to identify residues that might direct product specificity. The role and contribution of several of these residues to catalysis and product specificity were subsequently tested by the creation of site-directed mutants. One series of mutants was demonstrated to change the reaction product to a novel sesquiterpene, 4-epi-eremophilene, and while another series successfully transmutated TEAS into a HPS-like enzyme. This is the first report of a rational redesign of product specificity for any terpene synthase. The contact map provides a basis for the prediction of specific configurations of amino acids that might be necessary for as yet uncharacterized sesquiterpene synthases from natural sources. This prediction was tested by the subsequent isolation and validation that valencene synthase, a synthase from citrus, did indeed have the amino acid configuration as predicted. Lastly, an in vitro system was developed for analyzing the interaction between sesquiterpene synthases and the corresponding terpene hydroxylase. Development of this in vitro system is presented as a new important tool in further defining those biochemical features giving rise to the biological diversity of sesquiterpenes.

The biodiversity of Durban (eThekwini municipality) in KwaZulu Natal is primarily threatened by urbanization although other factors such as climate change and the spread of invasive species also pose a significant threat. Knowledge of what species exist within the city is important for biodiversity surveillance, detecting invasive taxa and uncovering cryptic species. Conducting a comprehensive biodiversity inventory is a daunting task, especially for hyperdiverse groups such as terrestrial arthropods, where closely related species can often only be separated by subtle morphological characters. This study investigated whether the barcoding marker, Cytochrome Oxidase C Subunit 1 (COI) can be used to efficiently and accurately delineate species of ants (family Formicidae) in comparison to traditional taxonomic approaches. The feasibility of DNA barcoding for assembling biodiversity inventories for urban areas which could be useful in conservation planning was also evaluated. A total of 619 individuals were sequenced from 23 geographic localities within the eThekwini region and surrounding regions. DNA barcoding revealed 80 provisional species/ “barcode clusters” or monophyletic lineages which could represent distinct species, while morphology revealed 51 different morphospecies. Extrapolation measures of species richness indicated that as many as 153 species of ants could occur in the city. Phylogenetic and phylogeographic analyses were performed on co-distributed species belonging to the genera Lepisiota, Camponotus, Pheidole and Pachycondyla to better understand the spatial distribution of genetic variability in the eThekwini region. Nuclear markers 18S rDNA and 28S rDNA were also sequenced and compared for a subsample of individuals from Camponotus and Pachycondyla. There was genetic variation at COI and the nuclear markers for each of the species examined. In order to fully elucidate the population genetic patterns which could be expected in eThekwini and surrounding regions, further sampling across more localities is essential. The use of more nuclear markers could also assist in uncovering these unique patterns of genetic variation in an urban setting. In this study, the utility of COI as a species diagnostic tool in ants was confirmed. The barcoding library constructed showed promise in highlighting reserves that should be preserved and possible cryptic speciation for further investigation. / M. Sc. University of KwaZulu-Natal, Durban 2014.

The purpose of the present study was to elucidate if caffeine ingestion reduced perception of effort at submaximal intensities during a maximal exercise test. A secondary purpose of this study was to examine the role of a single nucleotide polymorphism (SNP) at intron 1 of cytochrome P-450 gene in modulating caffeine’s influence on ratings of perceived exertion (RPE) at the same submaximal exercise intensities. Twelve healthy men (age: 24±1 yr., BMI: 23.9±1.2 kg.m2) volunteered to participate in the present study. Subjects consumed 6 mg.kg-1 of USP grade caffeine in 200ml of non-caloric, coloured and flavoured water, or a placebo-matched drink in a single-blind, randomised and crossover style design. Subjects remained seated for 1 hour after consuming the assigned drink, and subsequently completed an incremental maximal exercise test on a bicycle ergometer, which started at 0 Watts for 1 minute and increased by 25 Watts per minute until volitional exhaustion. RPE was reported every third minute during the test. DNA was obtained from whole blood samples and genotypes were determined using previously described methods. Similar to previous studies looking at this SNP, subjects were categorised into groups of AA homozygotes and C allele carriers for statistical analyses between genotypes. Two-way repeated measures ANOVA’s were performed (Treatment × Genotype) for RPE responses at submaximal workloads up to 300 Watts. Significant results were followed up using the bonferroni post-hoc method. There were no significant differences between individuals homozygous for the A variant and C allele carriers for age, height, weight, body mass index (BMI), and VO2max. A significant Time × Treatment interaction was observed (F=5.804, p<0.05) for the rate of increase in RPE between trials. A significant Treatment × Genotype interaction was also found (F=5.714, p<0.05), by which C allele carriers exhibited greater reductions in RPE during the caffeine trial compared to AA homozygotes. The findings of the present study indicate that perception of effort is reduced in individuals who metabolise caffeine at a slower rate (i.e. in C allele carriers). It is postulated that AA homozygotes do not experience reductions in RPE due to a greater cardiovascular workload and enhanced CNS excitability following caffeine ingestion / School of Physical Education, Sport, and Exercise Science

Rheumatoid Arthritis is a chronic inflammatory systemic disease and is one of the autoimmune diseases. In this study, nine candidate genes of the cytochrome P450 system have been analyzed to determine their possible association with the formation of RA. These genes are: CYP1A1, CYP1B1, CYP2B6, CYP2E1, CYP2C9, CYP2D6, CYP2A6, CYP2C19 and CYP3A4.Within these genes, 21 single nucleotide polymorphism, SNPs, in 300 French Caucasian individuals (100 RA trio families) were genotyped using single-base-extensions, SBE, in a mass spectrometric analysis by MALDI-TOF-MS (matrix-assisted Laser Desorption/ Ionization-time-of-flight mass spectometry). The selection of the examined genes was carried out taking into account known associations with RA or other autoimmune disease, as well as known functional variants. Decisive were also the location of the gene and genetic variability. The results of genotyping were used to study polymorphisms on their association with Rheumatoid Arthritis. The statistical analyzes of CYP2C9 rs1799853 (3011) showed, in the family-based single-marker test, a lower transmission (TDT p-value 0.021) for the rare allele (3011-a). The case-control allelic based test shows, there is a protective effect (Odds Ratio 0.58). In the case-control-based genotypic test this issue could be reproduced (p-value 0.046). For the rare allele of the CYP2A6 rs1801272 (3022-a) the family-based single-marker test shows a lower transmittance (TDT p-value 0.037). The case-control allelic based test shows a protective effect of this allele (Odds Ratio 0.32). In the case-control-based genotypic test a statistical trend to protective behavior of this allele occurs. SNPs with predicted functional relevance showed no statistical abnormalities in the studied cohort. In genome-wide studies, the results could not be tracked, at least at the gene level weak associations could be detected. The results of this study should be to replicate in one second independent cohort. Care should be taken specifically to xenobiotic stress, such as job stress, smoking, and medication. In subsequent studies more SNPs of the candidate genes could also genotyped in order to verify the genetic variability with reference to of the haplotypes in more detail. Should the above-mentioned associations be confirmed, functional studies on different gene expression or altered metabolite spectrum are highly interesting.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

Cytochromes c are electron transfer proteins in which haem is covalently attached to the polypeptide chain via thioether bonds formed from thiol groups of the two cysteines and the two vinyl groups of haem. This attachment is a post translational process and in many species of bacteria as many as approximately twelve gene products, the functions of which are largely unknown, are required. In Gram-negative bacteria the assembly of the c-type cytochromes occurs in the periplasm. Cytochrome c₅₅₂ from the thermophilic organism Hydrogenobacter thermophilus is known to be expressed in the cytoplasm of Escherichia coli. This unique example of cytoplasmic assembly of a c-type cytochrome has previously been postulated to result from insertion of haem into the folded apoform of the cytochrome followed by non-catalysed attachment of the haem. This postulate is supported by the present work which has shown that the cytoplasmic assembly of this cytochrome c₅₅₂ continues in the absence of the E. coli ccm genes which are needed for 'normal' c-type cytochrome assembly in that organism. Attempts to test the postulate of spontaneous assembly of the cytochrome c₅₅₂ with in vitro experiments require large amounts of cytochrome c₅₅₂ and its apo protein. A number of procedures for preparing these proteins were investigated. Although a T7-based expression produced lower amounts than was expected, its use led to detection of the apo form of cytochrome c₅₅₂ in E. coli. It was shown that this apoform has some secondary structure, whereas mitochondrial apocytochrome c has a random coil conformation. This observation is consistent with, but does not prove, the postulate for cytochrome c₅₅₂ assembly. It was unexpectedly found that a strain of E. coli that produces abnormally large amounts of its endogenous c-type cytochromes also made large amounts of cytoplasmic cytochrome c₅₅₂. NMR studies on this material are consistent with a single and 'normal' attachment of the haem to the polypeptide. Thus the unusual cytoplasmic assembly was not different from the usual periplasmic assembly that occurs in the H. thermophilus itself. In E. coli there is a periplasmic cytochrome b₅₆₂ that is presumed to acquire its haem in the periplasm. Some of the ccm genes, required for c-type cytochrome assembly, are postulated to code for a system that transports haem to the periplasm. Cytochrome b- ₅₆₂ synthesis was not blocked by the absence of these genes. This implies that haem provision for cytochrome b₅₆₂ synthesis occurs independently of the ccm system. Apocytochrome b<sub>562 could be detected in E. coli with the ratio apo:holo being higher in a strain that produces c-type cytochromes to relatively low levels. It is suggested that the synthesis of both cytochrome b₅₆₂ and c-type cytochromes is at least partly a reflection of the rate of production of haem by the cells.

Deficiency of the cytochrome P-450 steroid 21-hydroxylase (21-OHase) which causes Congenital Adrenal Hyperplasia (CAH) is a monogenic autosomal recessive disorder which is linked to HLA. There are two 21-OHase genes in man, A and B, and they are mapped to the HLA class III region ~ 3 kb 3' to the complement genes C4A and C4B, respectively. Two genes encoding 21-OHase were isolated, characterized and sequenced. Both 21-OHase genes are ~ 3.3 kb in length and are split into 10 exons by nine introns. Comparison of the two genes showed that although they are highly conserved, there are three deleterious mutations in the 21-OHase A gene which cause frameshifts and introduce in phase premature termination codons. Thus the 21-OHase A gene is a pseudogene. Comparison of the 21-OHase B gene to the other cytochrome P-450 sequences revealed that although the cysteine-429 was conserved in 21-OHase, there is very little homology with other cytochrome P-450, indicating it belongs to a separate family of genes within the superfamily. Clear evidence of polymorphism in 21-OHase is apparent on comparison with other 21-OHase B sequences. There is a size polymorphism of 494 and 495 amino acids. The differing severities of 21-OHase deficiency in CAH may be due to allelic variants of the 21-OHase B gene, since in most cases the defect is not due to gene deletion (Rumsby et al., 1986). A 21-OHase B gene from a patient with CAH was characterized and sequenced. There were 13 nucleotide alterations in his single 21-OHase B gene, one of which at codon 269 caused a serine to change to a threonine residue. The G → C transversion in the 21-OHase B gene from the patient at codon 269 introduced a new NcoI restriction site into the gene. This restriction fragment length polymorphism (RFLP) was used to study other patients with CAH and normal individuals. The NcoI RFLP was found not to be confined to the 21-OHase B gene but was also present in some 21-OHase A genes. It is likely therefore that the mutation occurred in the pseudogene first and then transferred to some 21-OHase B genes.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.