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 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.
Corrigall
A V
AV
MRC/UCT Liver Research Centre, University of Cape Town Medical School, Observatory, 7925, South Africa.
Siziba
K B
KB
Maneli
M H
MH
Shephard
E G
EG
Ziman
M
M
Dailey
T A
TA
Dailey
H A
HA
Kirsch
R E
RE
Meissner
P N
PN
D000332:Aerobiosis; D001412:Bacillus subtilis / Q000201:enzymology*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase
D004791:Enzyme Inhibitors; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase