PURIFICATION OF AND KINETIC-STUDIES ON A CLONED PROTOPORPHYRINOGEN OXIDASE FROM THE AEROBIC BACTERIUM BACILLUS-SUBTILIS

The previously cloned and expressed protoporphyrinogen oxidase from Ba cillus subtilis has been purified to homogeneity by Ni2+ affinity chro matography using a His, tag and characterized. The enzyme has a molecu lar 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 protoporphyr inogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5 .29, and 4.92 mu M, respectively. Polyclonal antibody to B. subtilis p rotoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herb icide acifluorfen at 100 mu M and is weakly inhibited by methylacifluo rfen at the same concentration. Bilirubin, biliverdin, and hemin are a ll competitive inhibitors of this enzyme. (C) 1998 Academic Press.