Mechanistic, structural and regulatory investigations of unusual and artificial variants of chorismate mutases

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Chorismate mutases (CM) of the Shikimate pathway are the only well-characterized enzymes that catalyze a pericyclic reaction. However, the exact nature of the catalytic mechanism and the high rate acceleration are still poorly understood. Especially the r elation between stabilizing charges versus catalytic center geometry is debated and needs to be clarified. Additionally, the activity of a new subgroup of CMs that is intrinsic to pathogens such as Mycobacterium tuberculosis is dependent on the first enzy me in the Shikimate pathway and the mode of regulation is still unclear. This project therefore aims on one hand to gain new insights into the catalytic mechanism by structurally and biochemically analyzing Bacillus subtilis CM variants with non-natural amino acids replacing a catalytic arginine. A mutant CM with an arginine-90-citrulline substitution is already available and exhibits extremely low activity. However, it is still able to bind chorismate, which facilitates the first structure determination of a CM with its natural substrate bound in place. We will also solve the structures of this CM variant with a transition state analog and the natural product, prephenate, bound in the catalytic center to definitely answer the question of the catalytic m echanism. On the other hand the new CM subclass from Mycobacterium tuberculosis will be structurally investigated in complex with the regulating enzyme, bound to its feedback regulators tyrosine and phenylalanine. Different active site mutations will b e structurally and enzymologically characterized, and those will also be the basis for virtual and high-throughput screening for inhibitor substances. We will also crystallize and structurally characterize the homologous enzymes of the related bacterium C orynebacterium glutamicum in order to determine if the regulation of this CM subclass by other Shikimate pathway enzymes is a general feature.