Expression and processing of human ornithine-δ-aminotransferase in Saccharomyces cerevisiae

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Abstract
Ornithlne-δ-aminotransferase catalyzes the conversion of omithine to glutamate-γ-semialdehyde. In humans, deficiency of this mitochondrial matrix enzyme results in the progressive blinding disorder, gyrate atrophy of the chorold and retina. To explore yeast as an expression system, we introduced a cDNA encoding human ornithine-δ-aminotransferase into an ornithine aminotransferase-deficient strain of Saccharomyces cerevlslae. 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-δ-aminotransferase in S.cerevislae is cytosolic, human ornithine-δ-aminotransferase expressed in S.cerevislae was localized to the mitochondrial matrix with correct proteolytic processing of its mitochondrial leader sequence. Despite this anomalous location in yeast, human omithine-δ-aminotransferase complemented the phenotype of the mutant strain, restoring its ability to utilize omithine as a sole nitrogen source. We also expressed a vitamin B6-responsive missense allele of ornithine-δ-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.

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