Expression and processing of human ornithine-delta-aminotransferase in Saccharomyces cerevisiae.
Ornithine-delta-aminotransferase catalyzes the conversion of ornithine to glutamate-gamma-semialdehyde. In humans, deficiency of this mitochondrial matrix enzyme results in the progressive blinding disorder, gyrate atrophy of the choroid and retina. To explore yeast as an expression system, we introduced a cDNA encoding human ornithine-delta-aminotransferase into an ornithine aminotransferase-deficient strain of Saccharomyces cerevisiae. The human enzyme was expressed at high levels, with activity 20-fold greater than that of wild-type yeast and 10-fold higher than in human fibroblasts. Although the normal location of ornithine-delta-aminotransferase in S. cerevisiae is cytosolic, human ornithine-delta-aminotransferase expressed in S. cerevisiae was localized to the mitochondrial matrix with correct proteolytic processing of its mitochondrial leader sequence. Despite this anomalous location in yeast, human ornithine-delta-aminotransferase complemented the phenotype of the mutant strain, restoring its ability to utilize ornithine as a sole nitrogen source. We also expressed a vitamin B6-responsive missense allele of ornithine-delta-aminotransferase (V332M) and showed that the biochemical phenotype of this allele is easily demonstrated confirming the usefulness of this system for examining mutations causing gyrate atrophy.
Dougherty
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Predoctoral Training Program in Human Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Swanson
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Brody
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Valle
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D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009953:Ornithine-Oxo-Acid Transaminase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic
D017931:DNA Primers; D011994:Recombinant Proteins; D009953:Ornithine-Oxo-Acid Transaminase