Tilbake til søkeresultatene

FRIMEDBIO-Fri prosj.st. med.,helse,biol

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

Tildelt: kr 3,1 mill.

Chorismate mutases (CMs) are enzymes that catalyze an essential reaction at the branching point of the metabolic shikimate pathway for the production of aromatic amino acids. As this pathway is confined to bacteria, fungi and plants, CMs therefore represent potential targets for highly specific antibiotics, fungicides or herbicides without adverse effects for animals and humans. Here, one should especially make note of Mycobacterium tuberculosis and related pathogens, whose CMs were also investigated in this project. In addition to these practical implications, CMs are the only well-characterized enzymes that catalyze a pericyclic reaction, and are therefore of significant interest to bioorganic chemists. So far, however, the exact nature of the catalytic mechanism and the high rate acceleration were still debated, especially the relative contributions of the transition state-stabilizing charges versus geometry in the catalytic pocket. We were able to elucidate the enzyme mechanism by structurally and biochemically analyzing a variant of the CM of Bacillus subtilis (BsCM), where the non-natural amino acid citrulline was used to replace an important arginine residue in the catalytic center, decreasing the activity more than 10000-fold. Citrulline, in which the guanidinium group of the arginine side chain is substituted with a urea group, is an uncharged analog of arginine, with the same shape and size. Therefore, with the rest of the active site being equal, the importance of the positive charge can be assessed. We solved the high-resolution crystal structures of this non-native CM variant in complex with substrate, transition state analog and product, as well as the apo structure, with resolutions ranging from 1.6 - 1.8 Å. The structures clearly show that the significant drop in enzymatic activity does not depend on changes in active site geometry but solely on the missing charge. Therefore the enzymatic mechanism proceeds via charge-mediated transition state stabilization and we were finally able to resolve the debate on this unusual class of enzymes. Using our high-resolution structures, we were able to reach a more detailed characterization of CM catalysis with a quantum mechanical computational approach. We aimed to compare the active sites of the wild type BsCM with Arg90Cit BsCM, to single out the effect of the charge difference on catalysis. Although this proved to be problematic, we were nonetheless able to assess the reaction semi-quantitatively. Interestingly, we did find small structural differences in fully optimized active site structures, which clearly show the competence of the wild type enzyme to stabilize transition state, while interacting much less strongly with the substrate. This finding suggests that the transition state analog, which is at the site of catalysis more similar to the substrate geometry than the transition state, is imperfect as an inhibitor, and could be improved. Regarding the CMs of Mycobacterium tuberculosis, we were able to reveal a new paradigm of Shikimate pathway regulation, based on the crystal structures of two Mycobacterium tuberculosis enzymes, plus allosteric regulators. Furthermore, we structurally characterized several enzyme variants, which were engineered for increased activity by directed evolution experiments. The corresponding wild-type enzymes from Corynebacterium glutamicum, which will be useful as a future model system, have also been successfully expressed and purified. The structure of CM in the inactivated state has been solved to 1.1 Å resolution, and the structure of DAHP synthase has been solved to 2.4 Å for the apo enzyme, and 2.8 Å with an inhibiting amino acid bound. Crystals of the two enzymes in complex with the transition state analog of CM have been produced, but structural analysis is pending.

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.

Budsjettformål:

FRIMEDBIO-Fri prosj.st. med.,helse,biol

Finansieringskilder