CLONING AND CHARACTERIZATION OF THE YEAST HEM14 GENE CODING FOR PROTOPORPHYRINOGEN OXIDASE, THE MOLECULAR TARGET OF DIPHENYL ETHER-TYPE HERBICIDES

Protoporphyrinogen oxidase, which catalyzes the oxygen dependent aroma tization of protoporphyrinogen IX to protoporphyrin IX, is the molecul ar target of diphenyl ether type herbicides. The structural gene for t he yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yea st mutant, using a novel one-step colored screening procedure to ident ify heme-synthesizing cells. The hem14-1 mutation was genetically link ed to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2. Disruptio n of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vit ro (lack of immunodetectable protein or enzyme activity). The HEM14 ge ne encodes a 539-amino acid protein (59,665 De; pi 9.3) containing an ADP-beta alpha beta-binding fold similar to those of several other fla voproteins. Yeast protoporphyrinogen oxidase was somewhat similar to t he HemY gene product of Bacillus subtilis and to the human and mouse p rotoporphyrinogen oxidases. Studies on protoporphyrinogen oxidase over expressed in yeast and purified as wild-type enzyme showed that (i) th e NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purifie d, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and r eadily photolabeled by a diazoketone derivative of tritiated acifluorf en. The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme. Both mutations introduced indepe ndently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coil expression system.