Folylpolyglutamate synthetase activities of Neurospora

Abstract

The folylpolyglutamate synthetase (FPGS) activities of Neurospora crassa, wild type (FGSC 853) and two polyglutamate-deficient mutants, met-6,35809 (FGSC 1330) and mac, 65108 (FGSC 3609), were examined after growth in defined media. Extracts of the wild type produced H 4PteGlu 6 (60 %), H 4PteGlu 3 (35 %) and H 4PteGlu 2 (15 %). Met-6 extracts formed H 4PteGlu 2 but lacked the ability to utilize H 4PteGlu 4 or H 4PteGlu 5. The mac mutant failed to catalyse glutamate addition to H 4PteGlu but H 4PteGlu 2 was an effective substrate for tri- and hexaglutamate synthesis. These polyglutamates were also formed by reaction systems containing mixtures of met-6 and mac protein or heterokaryon protein derived from mycelial fusions of met-6 and mac. Extract fractionations and heat treatments provided evidence for more than one FPGS activity in the wild type. A mitochondrial FPGS catalysed the H 4PteGlu 2 → H 4PteGlu 3 reaction but a cytosolic fraction synthesized di-, tri- and hexaglutamates when incubated with H 4PteGlu and glutamate. The latter system contained a temperature-sensitive diglutamate-forming activity and a relatively stable H 4PteGlu 2 → H 4PteGlu 6 activity. Polyglutamate synthesis in N. crassa appears to involve more than one step, H 4PteGlu → H 4PteGlu 2 followed by H 4PteGlu 2 → H 4PteGlu 6, in addition to the mitochondrial activity. These partial activities are lacking in mac and met-6 respectively. Consequently, these mutants are unable to form the folylhexaglutamates that predominate the folate pool of the wild type.