Purification of and Kinetic Studies on a Cloned Protoporphyrinogen Oxidase from the Aerobic BacteriumBacillus subtilis

The previously cloned and expressed protoporphyrinogen oxidase fromBacillus subtilishas been purified to homogeneity by Ni2+affinity chromatography using a His6tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pIof 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 μM, respectively. Polyclonal antibody toB. subtilisprotoporphyrinogen oxidase demonstrated weak cross-reactivity with both human andMyxococcus xanthusprotoporphyrinogen oxidase.B. subtilisprotoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 μM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.