Protoporphyrinogen Oxidase of Myxococcus xanthus

Abstract

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-␤-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 H 2 O 2 /mol of protoporphyrin. The apparent K m and k cat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 M and 5.2 min ؊1, respectively. The diphenyl ether herbicide acifluorfen at 1 M strongly inhibits the enzyme's activity.The penultimate step in the heme biosynthetic pathway, the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX, is catalyzed by the enzyme protoporphyrinogen oxidase (EC 1.3.3.4) (1-3). In eukaryotes this enzyme is located on the cytosolic side of the inner mitochondrial membrane and utilizes molecular oxygen as its terminal electron acceptor. The enzyme from two prokaryotes have been cloned, sequenced, expressed, and partially characterized. The enzyme from Bacillus subtilis (4, 5) is similar to the eukaryotic enzyme in that it contains a flavin and utilizes molecular oxygen as terminal electron acceptor. However, its substrate specificity is much broader than the eukaryotic enzyme since it will oxidize not only protoporphyrinogen IX, but also the pathway intermediate coproporphyrinogen III. In addition it is resistant to inhibition by the herbicide acifluorfen which strongly inhibits the eukaryotic enzyme. Protoporphyrinogen oxidase has also been cloned and expressed from Escherichia coli (6). The cloned protein is smaller in size than the B. subtilis and eukaryotic enzymes, does not contain an FAD binding motif, and is obligatorily coupled to the cell's respiratory chain. Based upon previous data from Desulfovibrio gigas (7) it would appear that the cloned E. coli enzyme is a subunit of a multi-protein complex.Data base searches using the B. subtilis protoporphyrinogen oxidase derived amino acid sequence yielded two similar bacterial sequences (8). In an effort to expand our knowledge about this enzyme and to discover if the herbicide resistance and broad substrate specificity found with the B. subtilis enzyme are characteristic of the prokaryotic, oxygen dependent enzymes, we expressed, purified, and characterized protoporphyrinogen oxidase of the...

Discovery and Characterization of HemQ

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