Browsing by Author "Meissner, Peter"

Now showing 1 - 9 of 9
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    Characterisation of the flavin adenine dinucleotide binding region in Myxococcus xanthus protoporphyrinogen oxidase
    (2011) Boateng, Mavis O; Meissner, Peter; Corrigall, Anne
    This dissertation focuses on protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway. Partial defects in PPOX result in variegate porphyria, an autosomal dominant disorder. PPOX catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen. FAD is a cofactor, functioning as an intermediate electron acceptor in the catalytic function of PPOX. In this study the FAD binding region in Myxococcus xanthus PPOX was analysed by engineering and characterising a selection of relevant mutants.
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    Expression, purification and characterisation of protoporphyrinogen oxidases from diverse species
    (2000) Siziba, Kwanele Bennett; Meissner, Peter; Corrigall, Anne
    This work involved the characterisation of protoporphyrinogen oxidase (PPO), the penultimate enzyme in haem biosynthesis, from Bacillus subtilis, Myxococcus xanthus, and human. A defect in human PPO causes variegate porphyria, an autosomal dominant disorder characterised by skin photosensitivity and propensity towards acute neurovisceral crises. At the beginning of this project little information was available on the kinetic and biophysical properties of isolated PPOs due largely to difficulties associated with their purification from natural sources.
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    Investigation of an atypical protoporphyric family in South Africa
    (2010) Haumann, Carel Eduard; Meissner, Peter
    Affected members of the family investigated in this dissertation presented with photosensitivity and raised red cell protoporphyrin concentrations, indicative of protoporphyria. Further examination of this family revealed features that were atypical of erythropoietic protoporphyria. These included a highly penetrant disease, disease severity as expressed by more prevalent hepatic complications, a preponderance of protoporphyrin in its zinc chelated form, a therapeutic response to iron supplementation, and an absence of mutations in the ferrochelatase gene or haplotype markers associated with erythropoietic protoporphyria. We have reviewed clinical data from this family, established a ferrochelatase enzyme assay in our laboratory, and shown normal ferrochelatase enzyme activity in affected subjects.
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    Mitochondrial targeting of wild-type and mutant human protoporphyrinogen oxidase (PPOX)
    (2003) Davids, Lester M; Meissner, Peter; Corrigall, Anne
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    Molecular and Kinetic Characterisation of wild type and mutant Porphobilinogen Deaminase
    (2016) Pienaar, Elaine; Meissner, Peter
    The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.
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    Molecular characterisation of acute intermittent porphyria in South Africa
    (2014) Fortgens, Philip Hendrik; Meissner, Peter; Corrigall, Anne; Berman, Peter; Pillay, Tahir
    Acute intermittent porphyria belongs to a group of inherited disorders of haem metabolism. The object of this project is to characterise the mutations in the hydroxymethylbilane synthase (HMBS) gene in a cohort of South African patients. The elucidation of these mutations will facilitate an understanding of the molecular basis of AIP in South Africa, and provide a platform for the screening of family members of affected patients. Identification of latent carriers would allow for education with respect to precipitants and how best to avoid them, so as to minimise the risk of provoking an acute attack.
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    Molecular characterisation of erythropoietic protoporphyria in South Africa
    (2006) Parker, Michelle; Meissner, Peter; Corrigall, Anne; Hift, Richard
    Includes bibliographical references.
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    Studies on human protoporphyrinogen oxidase
    (2002) Maneli, Mbulelo H; Meissner, Peter; Corrigall, Anne
    This study examines the effects of various protoporphyrinogen oxidase mutations responsible for variegate porphyria, the role of the arginine-59 residue, and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding. Wild type recombinant human protoporphyrinogen oxidase and a selection of both naturally occurring and self-designed mutants were generated, expresses and purified. The self designed mutants included a conservative and two non-conservative arginine-59 replacements, and substitution of glycine residues at positions 9, 11, and 14 by alanine. The expression and purification for all protoporphyrinogen oxidases was optimised, enabling their purification to homogeneity by single step metal affinity chromatography.
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    A study of aminolaevulinic acid synthase
    (2001) Nahman, Carolyn Joy; Kirsch, Ralph; Meissner, Peter
    Delta-aminolaevulinic acid synthase (ALAS) is the first enzyme in the haem biosynthetic pathway, and in mammals is present in two isoforms - differing in regulation and encoded for on different chromosomes. The hepatic form is up-regulated in states of haem deficiency and thus implicated in the acute attack of porphyria. Given the large number of patients who suffer from an acute form porphyria in South Africa the regulation of the human hepatic form of the enzyme is of considerable interest.