CHARACTERIZATION OF THE SACCHAROMYCES-CEREVISIAE ARG7 GENE ENCODING ORNITHINE ACETYLTRANSFERASE, AN ENZYME ALSO ENDOWED WITH ACETYLGLUTAMATE SYNTHASE ACTIVITY

We have cloned by functional complementation and characterized the yea st ARG7 gene encoding mitochondrial ornithine acetyltransferase, the e nzyme catalyzing the fifth step in arginine biosynthesis. While formin g ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interest ingly, total deletion of the genomic ARG7 ORF resulted in an arginine- leaky phenotype, indicating that yeast cells possess an alternative ro ute for generating ornithine from acetylornithine. Yeast ornithine ace tyltransferase has been purified and characterized previously as a het erodimer of two subunits proposed to derive from a single precursor pr otein [Liu, Y.-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) E ur. J. Biochem. 228, 291-296]; those authors further suggested that th e internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cle avable-type. The characterization of the gene (a) establishes that Arg 7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondri al proteolytic peptidase target sequence. Yeast Arg7p shares between 3 2-43% identity in pairwise comparisons with the ten analogous bacteria l ArgJ enzymes characterized. Among these evolutionarily related enzym es, some but not all appear bifunctional, being able to produce acetyl glutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutam ate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylgluta mate synthase). Furthermore, Arg7p-linked acetylglutamate synthase act ivity was measurable in an assay. The yeast enzyme is thus clearly, al beit modestly, bifunctional. As with several bacterial ornithine acety ltransferases, the activity of Arg7p was practically insensitive to ar ginine but strongly inhibited by ornithine, which behaved as a competi tive inhibitor.