The Regulation of Methionine Synthesis and the Nature of Cystathionine γ-Synthase in Neurospora

Abstract

Abstract Methionine synthesis in Neurospora involves acetylation of homoserine, catalyzed by homoserine transacetylase; replacement of the acetyl group by cysteine, catalyzed by cystathionine γ-synthase; and elimination of homocysteine from cystathionine, catalyzed by β-cystathionase. Another enzyme catalyzes reactions of acetylhomoserine with sulfide or methylmercaptan to yield homocysteine or methionine directly, but the acetylhomoserine sulfhydrylase reaction does not play a major role in methionine synthesis in this organism. Three other fungi, Saccharomyces, Candida, and Cunninghamella were found to have transacetylase and sulfhydrylase enzymes similar to those in Neurospora, but only equivocal levels of cystathionine synthase have so far been detected. The Neurospora cystathionine synthase activity was very labile after extraction and was lost on dilution. Inhibition by hydroxylamine could be only partially attributed to a requirement for pyridoxal phosphate. Eight genes are known in Neurospora in which mutation confers methionine auxotrophy. None of the corresponding mutants lacked the sulfhydrylase. The me-5 mutants investigated had modified, rather than absent, transacetylase activities. Cystathionine synthase activity was absent from extracts of me-3, me-7, me-1, and me-6 mutants. The first two of these mutants have no other known defect; the second two are also blocked in the transmethylation of homocysteine. Activity was reconstituted by pairwise mixing of macromolecular fractions from the four mutant extracts in all combinations, with one possible exception, suggesting that several gene products are involved in this presumably single step reaction. Addition of methionine to minimal growth medium did not affect the wild type levels of transacetylase or cystathionine synthase activity. S-Adenosylmethionine was identified as an end product inhibitor of cystathionine synthase. Activity was inhibited 50% by 0.01 mm S-adenosylmethionine, but the inhibition was established only after the enzyme had been exposed to inhibitor for 5 min. No end product control over the first reaction unique to methionine synthesis could be detected, suggesting the existence of another, unidentified vital function for acetylhomoserine, or for the transacetylase. The concentration of S-adenosylmethionine in wild type cells which had been grown in the presence of methionine was shown to be sufficient to inhibit cystathionine synthase completely. An ethionine-resistant mutant which over-produced methionine was found to be unable to accumulate the inhibitor due to a defect in S-adenosylmethionine synthase; this is believed to be the first identification of a gene coding this enzyme.