[show abstract][hide abstract] ABSTRACT: The effects of various protoporphyrinogen oxidase (PPOX) mutations responsible for variegate porphyria (VP), the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant human PPOX and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The K(m)s for the arginine-59 mutants suggested a substrate-binding problem. T(1/2) indicated that arginine-59 is required for the integrity of the active site. The dominant alpha-helical content was decreased in the mutants. The degree of alpha-helix did not correlate linearly with T(1/2) nor T(m) values, supporting the suggestion that arginine-59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two-state transition suggesting formation of a multi-domain structure. All mutants studied were more resistant to thermal denaturation compared to wild type, except for R168C.
Biochimica et Biophysica Acta 09/2003; 1650(1-2):10-21. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 microM, respectively. Polyclonal antibody to B. subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 microM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.
Archives of Biochemistry and Biophysics 11/1998; 358(2):251-6. · 3.37 Impact Factor