Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.
Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.
Dailey
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Department of Microbiology, University of Georgia, Athens 30602-2605, USA. dailey@bscr.uga.edu
Dailey
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005420:Flavoproteins; D006801:Humans; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005420:Flavoproteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase