PROTOPORPHYRINOGEN OXIDASE OF MYXOCOCCUS-XANTHUS - EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CLONED ENZYME

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxi dation 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 apparen t homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per Liter of culture, Based upon the deduced amino ac id sequence the molecular weight of a single subunit is 49,387, Gel pe rmeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopy ranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000, The native enzyme i s, thus, a homodimer, The purified protein contains a non-covalently b ound FAD but no detectable redox active metal, The BL xanthus enzyme u tilizes protoporphyrinogen IX, but not coproporphyrinogen III, as subs trate and produces 3 mol of H2O2/mol of protoporphyrin, The apparent K -m and k(cat) for protoporphyrinogen in assays under atmospheric conce ntrations of oxygen are 1.6 mu M and 5.2 min(-1), respectively, The di phenyl ether herbicide acifluorfen at 1 mu M strongly inhibits the enz yme's activity.