Studies on Normal and Genetically Altered Tryptophan Synthetase from Neurospora Crassa.

Reaction (1) has been studied in some detail in extracts from Neurospora crassa.6 This reaction requires the presence of pyridoxal phosphate. Further studies demonstrated that extracts from wild type N. crassa also catalyze reactions (2) and (3), and suggested that InGP is converted to tryptophan by wild type N. crassa without the formation of free indole.2 3 While a majority of N. crassa auxotrophs which lack tryptophan synthetase (td mutants) accumulate indole glycerol in their growth filtrate, two accumulate indole.6 If the final step in tryptophan biosynthesis is the conversion of InGP to tryptophan, the accumulation of indole by these td mutants cannot be explained by the absence of a single enzyme. The following data indicate that the accumulation of indole by td mutants is attributable to the presence of an altered tryptophan synthetase, which catalyzes reaction (3) but not reactions (1) or (2). Materials and Methods. Mutant td 2, which was previously studied,7 and td 71, which was obtained by X-irradiation of the wild type strain 74A,6 were used. Both of these mutants require tryptophan for growth and accumulate indole in their culture filtrates, while all other td mutants which have been isolated accumulate indole glycerol in their growth medium. Both td 2 and td 71 have been shown to be allelic with other td mutants.6 Wild type 74A, which has been used for these studies, was grown on minimal medium8 and mutant strains td 2 and td 71 were grown on minimal medium plus 100 ,ug L-tryptophan per ml. Cultures were grown on a rotary shaker at 30°C for 48-72 hours. Mycelia were filtered through cheesecloth, washed and lyophilized. The lyophilized mycelia were powdered in a Wiley mill and stored at 15°C. Extracts were prepared at 4°C by grinding one part powdered, lyophilized mycelia with two parts alumina A-301 with the gradual addition of 15 parts of 0.05 M phosphate buffer, pH 7.8. The extract was centrifuged at 100,000 X g for 30 min and the supernatant used as the crude extract. The crude extract always contained 15-20 mg of protein per ml. Fractionation procedures were carried out for either concentration or purification of enzyme activity and were the same for both wild type and mutant strains. To