Biosynthesis of Aromatic Amino Acids in Yeast and Aspergillus

Abstract

Chorismate mutases (E.C. 5.4.99.5) catalyse the Claisen rearrangement of chorismic acid to prephenate, the first reaction of the tyrosine/phenylalanine-specific branch of aromatic amino acid biosynthesis. Within this biosynthetic pathway they compete with the anthranilate synthase complex for the common substrate chorismate and both enzymes define the first branch point of that metabolic pathway. The ARO7-encoded chorismate mutase of the bakerís yeast Saccharomyces cerevisiae which has previously been characterized in detail serves as model system for allosteric regulation of catalytic activity. Other chorismate mutases of fungi are hardly characterized. In this thesis, the mechanisms regulating the enzymatic activities that channel chorismate into the two main branches in S. cerevisiae were analysed. The impact of an allosterically unregulated chorismate mutase was investigated in combination with genetically engineered variations in transcriptional regulation of expression of both branch point genes. It turned out that only the regulatory pattern as it exists at the branch point is sufficient under amino acid starvation conditions. The results imply that in the bakerís yeast transcriptional and allosteric regulation have evolved in accordance to guarantee optimal flux of the intermediate compound into both branches. For comparison, the chorismate mutase activity of a methylotrophic yeast, the HARO7 gene product of Hansenula polymorpha, was characterized. The allosteric enzyme is strictly regulated by the end products tyrosine and tryptophan. In the presence of methanol as sole carbon source, transcription of the encoding gene is induced, whereas under amino acid starvation conditions no additional transcriptional regulation is present. In order to characterize the chorismate mutase of a filamentous fungus, the aroC-encoded enzyme of Aspergillus nidulans was investigated. Here, again no transcriptional regulation upon starvation conditions is present, but allosteric regulation by the heterotropic effectors tyrosine and tryptophan. Catalytic properties of the gene product were determined as well as its quaternary structure. Furthermore, it was shown that the allosteric intramolecular signal transduction pathway is not conserved with respect to the bakerís yeast enzyme.