The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated chorismate mutase

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 e ncoding chorismate mutase (EC 5.4.99.5), the enzyme at the first branch poi nt of aromatic amino acid biosynthesis, The aroC gene complements the Sacch aromyces cerevisiae aro7 Delta as well as the A. nidulans aroC mutation. Th e gene consists of three exons interrupted by two short intron sequences. T he expressed mRNA is 0.96 kilobases in length and aroC expression is not re gulated on the transcriptional level under amino acid starvation conditions , aroC encodes a monofunctional polypeptide of 268 amino acids. Purificatio n 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 o f 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative a cting, heterotropic feedback-inhibitor with a K-i of 2.8 mu M. Immunologica l 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(23 3)) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity.