Genes and isoenzymes controlling the first step in the aromatic amino acid biosynthesis in Schizosaccharomyces pombe

Abstract

1. 1. Crude extracts of Schizosaccharomyces pombe wild-type were assayed in order to characterize the first enzyme of the aromatic amino acid pathway, 3-deoxy- d- arabino-heptulosonate-7-phosphate synthase (7-phospho-2-keto-3-deoxy- d- arabino-heptonate- d-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase). Optimal conditions for activity have been found with a 0.1 M phosphate buffer (pH 6.5). Co 2+ and Mn 2+ stimulate activity whereas EDTA inhibits strongly. Inhibition by EDTA is reversed by Co 2+. The enzyme activity is feedback inhibited by tyrosine and phenylalanine but not by tryptophan. 2. 2. On analyzing the inhibition pattern in the activity peak obtained by Sephadex G-100 filtration, two overlapping regions with different degrees of inhibition by phenylalanine and tyrosine were found. 3. 3. DAHP synthase mutants were obtained and characterized. Aro2-C mutants show a reduced growth on minimal medium containing phenylalanine (1 mM) and aro1-C mutants grow poorly on minimal medium supplemented with tyrosine. In vitro the DAHP synthase activity of aro2-C mutants is inhibited to a greater extent by phenylalanine than in the wild-type, and aro-C mutants are more inhibited by tyrosine compared to the wild-type. 4. 4. Linkage relationships assign all aro1-C mutants to one locus, and all aro2-C mutants to another locus. The two genes are unlinked. 5. 5. Double mutants obtained from aro1-C × aro2-C crosses require tyrosine, phenylalanine, tryptophan and 4-aminobenzoate for growth. They do not grow on minimal medium or minimal medium supplemented with only one or with any combination of two aromatic amino acids plus 4-aminobenzoate. In vitro they show no DAHP synthase activity. 6. 6. These results show that in S. pombe the first step in the biosynthesis of aromatic amino acids is catalyzed by two isoenzymes.