DAHP Synthasen aus Pilzen

Abstract

The first step of the biosynthesis of the aromatic amino acids is the condensation of erythrose-4-phosphate and phosphoenolpyruvate to 3-deoxy-D-arabino-heptuloso-nate 7-phosphate. This reaction is catalyzed by the DAHP synthase (EC 4.1.2.15). There is an isoenzymatic system with two or three enzymes, dependant on the organism, which are either unregulated or inhibited by the end products phenylalanine, tyrosine or tryptophan. The yeast Saccharomyces cerevisiae carries two DAHP synthases, which are inhibitable by phenylalanine or tyrosine. Beside these two regulatory mechanisms E. coli carries a third isoenzyme which is inhibitable by tryptophan. The heterologous expression of this E. coli enzyme in yeast is stable. The gene product is able to rescue a Dgcn4 yeast strain which is not able to activate its general control when inhibited by high amounts of tyrosine and phenylalanine. Random mutagenesis as well as site directed mutagenesis experiments showed that residues are involved in the regulation of the tryptophan-inhibitable DAHP synthase which are not important for regulation of the two other isoenzymes from E. coli. In the filamentous growing fungus Aspergillus nidulans two DAHP synthases were identified and characterized. The two new isoenzymes are inhibitable by phenylalanine or tyrosine and carry a bivalent metal ion as cofactor. In contrast to the all known DAHP synthases both A. nidulans isoenzymes are not completely inhibitable by EDTA. Between these two DAHP synthases there a fine-tuning in regulation exists, which enables the fungus to control the carbon-flux into the shikimate pathway in a very sensitive way. To understand the function of the DAHP synthase in a more detailed way the protein structure of the tyrosine-inhibitable isoenzyme of the yeast Saccharomyces cerevisiae was solved. It was found to be a TIM barrel fold with some extra parts. Various mutagenesis experiments revealed that these extra parts are crucial for the regulation of this isoenzyme. To exchange the regulation properties of both yeast isoenzymes it needs only one amino acid, glycine 226 (serine in the respectively isoenzyme).