The cDNA and the chromosomal locus of the aroC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding chorismate mutase (EC 5.4.99.5), the enzyme at the first branch point of aromatic amino acid biosynthesis. The aroC gene complements the Saccharomyces cerevisiae aro7Delta as well as the A. nidulans aroC mutation. The gene consists of three exons interrupted by two short intron sequences. The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions. aroC encodes a monofunctional polypeptide of 268 amino acids. Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k(cat) = 82 s(-1), n(H) = 1. 56, [S](0.5) = 2.3 mM), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with a K(i) of 2.8 microM. Immunological data, homology modeling, as well as electron microscopy studies, indicate that this chorismate mutase has a dimeric structure like the S. cerevisiae enzyme. Site-directed mutagenesis of a crucial residue in loop220s (Asp(233)) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.

• PMID: 10428795
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Comparative Study | Journal Article | Research Support, Non-U.S. Gov't

Authors

Krappmann S, Helmstaedt K, Gerstberger T, Eckert S, Hoffmann B, Hoppert M, Schnappauf G, Braus GH

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