Regulative fine-tuning of the two novel DAHP isoenzymes aroFp and aroGp of the filamentous fungus Aspergillus nidulans

Abstract.

Two novel genes, aroF and aroG, from the filamentous fungus Aspergillus nidulans were isolated and the regulative fine-tuning between the encoded, differentially regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases was analyzed. A wide range of DAHP synthase isoenzymes of various organisms are known, but only a few have been characterized further. DAHP synthases (EC 4.1.2.15) catalyze the first committed step of the shikimate pathway, which is a putative target for anti-weed drugs. The reaction is the condensation of erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to yield DAHP. The two purified DAHP synthases showed different affinities for the substrates: 175 µM for PEP and 341 µM for E4P for the aroFp isoenzyme and weaker affinities of 239 µM (PEP) and 475 µM (E4P) for the aroGp isoenzyme. The enzymes are differentially regulated by tyrosine (aroFp) and phenylalanine (aroGp). The calculated k _cat values are 7.0 s^–1 for the tyrosine-inhibitable (aroFp) and 5.5 s^–1 for the phenylalanine inhibitable (aroGp) enzyme. Tyrosine is a competitive inhibitor of the aroFp DAHP synthase in its reaction with PEP. Phenylalanine is a competitive inhibitor of the isoenzyme aroGp in its reaction with E4P. Both enzymes are inhibited by the chelating agent EDTA, which indicates a metal ion as cofactor.