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Camadro JM, Labbe P: Cloning and characterization of the yeast HEM14 gene coding for protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. J Biol Chem. 1996 Apr 12;271(15):9120-8.
Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX, is the molecular target of diphenyl ether type herbicides. The structural gene for the yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yeast mutant, using a novel one-step colored screening procedure to identify heme-synthesizing cells. The hem14-1 mutation was genetically linked to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2. Disruption of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vitro (lack of immunodetectable protein or enzyme activity). The HEM14 gene encodes a 539-amino acid protein (59,665 Da; pI 9.3) containing an ADP- beta alpha beta-binding fold similar to those of several other flavoproteins. Yeast protoporphyrinogen oxidase was somewhat similar to the HemY gene product of Bacillus subtilis and to the human and mouse protoporphyrinogen oxidases. Studies on protoporphyrinogen oxidase overexpressed in yeast and purified as wild-type enzyme showed that (i) the NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purified, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and readily photolabeled by a diazoketone derivative of tritiated acifluorfen. The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme. Both mutations introduced independently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coli expression system. |
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