Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis

The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His, 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 (K-m) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 mu M, 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 mu M and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme. (C) 1998 Academic Press.