Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme.
Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.
Dailey
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Department of Microbiology, University of Georgia, Athens, 30602-2605, USA.
Dailey
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016941:Myxococcus xanthus / Q000201:enzymology*; D009579:Nitrobenzoates / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D017931:DNA Primers; D009579:Nitrobenzoates; D011994:Recombinant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; C018425:acifluorfen