Regulative fine-tuning of the two novel DAHP isoenzymes aroFp and aroGp of the filamentous fungus Aspergillus nidulans
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O R I G I N A L PA P E R
Markus Hartmann · Gabriele Heinrich · Gerhard H. Braus
Regulative fine-tuning of the two novel DAHP isoenzymes aroFp and aroGp of the filamentous fungus Aspergillus nidulans
Received: 4 September 2000 / Revised: 4 November 2000 / Accepted: 22 November 2000 / Published online: 2 February 2001 © Springer-Verlag 2001
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 kcat 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. Keywords Aspergillus nidulans · Protein purification · Enzyme kinetics · Regulation · 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthase Abbreviations DAHP 3-Deoxy-D-arabino-heptulosonate7-phosphate synthase · PEP phosphoenolpyruvate · E4P erythrose-4-phosphate
M. Hartmann · G. Heinrich · G. H. Braus (✉) Institute for Microbiology and Genetics, Georg August University, Grisebachstrasse 8, 37077 Göttingen, Germany e-mail: [email protected], Tel.: +49-551-393770
Introduction The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase) catalyzes the first step of the aromatic amino acid biosynthetic pathway, i.e., the condensation of phosphoenolpyruvate (PEP) and erythrose-4phosphate (E4P) to DAHP and inorganic phosphate (Haslam 1993). A number of DAHP synthase isoforms, distinguished according to their regulatory properties, exist in microorganisms and plants (Byng et al. 1982; Byng and Jensen 1983). The only known DAHP synthases from filamentous fungi are the three DAHP synthase isoforms of Neurospora crassa. Each is regulated by one of the following three aromatic amino acids: phenylalanine, tyrosine, and tryptophan (Hoffman et al. 1972; Nimmo and Coggins 1981a, b). Only one of the three isoforms, the tryptophan-inhibitable protein, has been analyzed further. Although all DAHP synthases catalyze the same reaction, they differ in size and mode
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