Chorismate mutase (EC 188.8.131.52) plays an important role for the biosynthesis of aromatic amino acids. Located at the branchpoint of the shikimate pathway, this enzyme channels amino acid precursors to the biosynthesis of phenylalanine and tyrosine and away from that of tryptophan. In many organisms, chorismate mutase is important for regulating the balance of aromatic amino acids in the cell.
The *MtCM protomer (PDB entry 2FP2; representative of the AroQg subclass).
About the project
Since the shikimate pathway and hence chorismate mutase only exist in bacteria, fungi and higher plants, the enzyme has a strong potential for the development of herbicides as well as anti-bacterial and anti-fungal therapeutics. Chorismate mutase catalyzes the rearrangement of chorismate to prephenate. This reaction is formally a Claisen rearrangement. Chorismate mutase is the only well-characterized enzyme that catalyzes a pericyclic process and has thus generated considerable interest in the bioorganic circles. Despite extensive studies, however, the enzyme mechanism and in particular the million-fold rate acceleration by chorismate mutase has remained poorly understood.
The crystallographic analysis of wild type and mutant chorismate mutase from B. subtilis and its complexes with various inhibitors and prephenate is an ongoing research project, which was started about twenty years ago at Harvard University, Cambridge, Massachusetts.
My first contribution to this study was the analysis of four crystal structures of different mutants at Arg 90, a residue indispensible for the activity of the enzyme. Two single mutants (R90G and R90A) showed no measurable enzyme activity, while two double mutants (R90KC88S and R90SC88K) regained activity due to a second mutation at Cys 88. This investigation strongly suggested that a strategically positioned cation is crucial for efficient catalysis by chorismate mutases (Kast et al., 2000).
The active site of *MtCM. The bound transition state analog inhibitor is highlighted with orange carbon atoms. Hydrogen bonds are indicated by dotted lines.
Recently, we have extended our investigation to include the two chorismate mutases of Mycobacterium tuberculosis. In contrast to the B. subtilis enzyme, which adopts the trimeric β-fold typical for AroH class chorismate mutases, the two M. tuberculosis enzymes belong to the AroQ class of chorismate mutases, which exhibit an all a-helical fold. We have recently solved the crystal structures of both M. tuberculosis enzymes (Ökvist et al., 2006 and Sasso et al., 2009). The secreted enzyme (*MtCM) exhibits an interesting new fold topology explaining the extreme stability of this enzyme, while the internal, house-keeping enzyme (MtCM) is more similar in structure to the AroQ prototype enzyme from Escherichia coli, EcCM. While MtCM has rather low activity on its own, its catalytic efficiency increases >100-fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate-pathway enzyme (Sasso et al., 2009). The structure of the MtCM-MtDS complex suggests how key active site residues of MtCM are repositioned upon interaction with MtDS, to rescue its activity (Sasso et al., 2009).
As it turns out, the chorismate mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature, which we are set to explore. - Stay tuned!
This project involves the close collaboration with Peter Kast’s research group at the ETH in Zürich, Switzerland.