Expression, purification and characterisation of protoporphyrinogen oxidases from diverse species
Year: 2000
Type: dissertation
Abstract: Expression, Purification and Characterisation of Protoporphyrinogen Oxidases from Diverse SpeciesThis 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.Recombinant PPOs of B. subtilis, M xanthus and human, expressed in E. coli cells were purified to apparent homogeneity by a rapid metal chelate affmity chromatography procedure.These PPOs with subunit molecular weights ranging from 52000 to 54000 Da were shown to be F AD containing flavoproteins.Optimal oxidation of the substrate, protoporphyrinogen-IX by the B.subtilis and M xanthus PPOs occurred at pH 8.7 and 8.1, respectively.Whilst both the human and M xanthus PPOs were highly specific for protoporphyrinogen-IX as substrate, the B. subtilis enzyme utilised both mesoporphyrinogen-IX and coproporphyrinogen-III as well.The fatty acids, oleic and palmitic, stimulated M xanthus PPO activity, whereas the B. subtilis enzyme was inhibited.Inhibition studies enabled us to establish the inhibitor profiles of these PPOs.Acifluorfen and methylacifluorfen, which strongly inhibited both human and M xanthus PPOs were ineffective against the B. subtilis enzyme.Human PPO was weakly inhibited by hemin and biliverdin, which strongly inhibited both B. subtilis and M xanthus enzymes.At 100 J.lM bilirubin did not inhibit human and M xanthus PPOs, whilst approximately 95% inhibition of the B. subtilis enzyme was observed.This revealed interspecies differences in the behaviour of PPOs towards various compounds.In all cases effective inhibitors produced a competitive mode of inhibition with respect to protoporphyrinogen-IX.Phylogenetic analysis using 28 different PPO protein sequences was perf?rmed, and interesting divergences amongst the prokaryotic forms of this protein were revealed.Immunocharacterisation studies with a polyclonal antibody raised against human PPO were used to demonstrate the differential expression of PPO in various human tissues/organs.Immunogold labelling and electron microscopy studies on human hepatocytes confirmed predominant location of PPO in mitochondria and also demonstrated cytosolic forms of PPO in these cells.
Author: Kwanele Bennett Siziba
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2230096297