Yeast Chorismate Mutase: Molecular Evolution of an Allosteric Enzyme

Abstract

Chorismate mutase (CM, EC 5.4.99.5), encoded by ARO7, catalyzes the Claisen rearrangement of chorismate to prephenate in the biosynthesis of the amino acids tyrosine and phenylalanine. The small, dimeric enzyme of the yeast Saccharomyces cerevisiae is allosterically activated by tryptophan and allosterically inhibited by tyrosine. In this work, earlier data in the literature which suggested that chorismate mutase functions in osmoregulation and vacuole biogenesis were disproven. The analysis of several strains containing aro7 point mutations or deletions did not show any other function for CM but its role in amino acid biosynthesis. By fusion to the green fluorescent protein, this protein was localized in the cytoplasm as well as in the nucleus.On the protein level, the intramolecular signal transduction from the allosteric to the active sites occuring upon effector binding was investigated in more detail. Chimeric enzymes were constructed, in which the molecular hinge loop L220s connecting the allosteric and catalytic domain in the dimer interface, were subtituted by the corresponding loops from homologous fungal enzymes. Kinetic analysis verified that this structural component is critical for protein stability and distinguishes between the activation and inhibition signal. This hinge is also involved in dimerization of the protein. Substitution of hydrophobic amino acids in and near this loop by charged residues produced a stable monomeric enzyme variant. This chorismate mutase showed reduced activity and lost allosteric regulation, but the encoding gene complemented phenylalanine and tyrosine auxotrophy of an aro7 mutant strain. These results supported the theory that yeast CM originated from a monomeric, unregulated ancestral protein similar to the Escherichia coli CM by coevolution of regulatory and stabilizing elements.In order to gain further insight into the principles of protein stabilization, the chorismate mutase from Thermus thermophilus was purified and analyzed after cloning of the structural gene aroG. This enzyme was similar to the structurally unique CM from Bacillus subtilis, but in contrast to the latter CM was inhibited by tyrosine. Computer modeling studies revealed that like in other proteins enhanced hydrophilicity on the protein surface, increased hydrophobicity of residues within the tertiary structure as well as the tightening of active site loops stabilized the protein fold.