Discussion

Structural analysis on M. tuberculosis chorismate synthase was carried out using X-ray crystallographic studies combined with molecular dynamics (MD) simulations.

High resolution structures of the enzyme in unliganded and liganded states and comparison to a highly homologue crystal structure allowed for the first time the detailed description of a ligand binding cycle that is suggested to be generally applicable to CS (Fig. 4-10). This binding cycle describes all the key conformational events associated with ligand binding to chorismate synthase and describes the role of several invariant residues present in all CS. Notably, the presented structural data point to the dependency of all key events of sequential ligand on conformational changes of a special invariant arginine (Arg341 in MtCS and Arg337 in SpCS; see also Fig. 4-4). This arginine is of clear necessity for the correct enzymatic mechanism by fine-tuning the active site for catalysis during formation of contacts to EPSP (Fig.

4-11b/c). Further investigations with the help of MD simulations clearly underlined the importance of this residue for the formation of a catalytically competent ternary enzyme complex. The role of the invariant arginine 341 in MtCS is of special importance considering a novel ligandation state with nicotinamide that this work presents for the first time. Structural data indicate that the binding mode of NCA might support an unproductive conformation of the active site in the apo form that involves Arg341 and therefore a side route to the formation of the binary complex is provided. However, the possibility exists to replace NCA by FMN in MtCS crystals by soaking (Fig. 4-10b/d).

Yet, the observed formation of a stable MtCS-NCA complex may be of further interest considering the known antimicrobial activity of nicotinamide [Chorine V, 1945]. NCA previously was used as an anti-TB drug, until antagonism between NCA and the first-line drug isoniazid was noticed [Murray MF, 2003]. Combined treatment with NCA and the analogue pyrazinamide (PZA), another first-line drug, may cause cross-resistance [Somoskovi A et al., 2001] due to the fact that both compounds are substrates of the mycobacterial enzyme nicotinamidase/pyrazinamidase (PZAse), which converts both prodrugs into the active forms nicotinic acid and pyrazinoic acid (POA). Recently, evidence has been provided for anti-HIV activity of nicotinamide, suggesting its potential use both as an antimicrobial and an antiretroviral agent [Murray MF, 2003]. However, the mechanism of the antimicrobial action of nicotinamide has not been elucidated until now. The observation presented in this work that NCA firmly binds to chorismate synthase suggests that this influence on the shikimate pathway of M. tuberculosis may be part of the antimicrobial activity of NCA.

Notably, the MtCS-NCA complex displayed substantially higher stability in acid medium (pH ~ 5) than at conditions near neutral pH, as shown by the crystallization studies for this work. Notably, acidic pH characterizes the environment of mycobacteria in macrophages. Similarly it has been shown that the analogous compound PZA requires acidic pH values to be active against M. tuberculosis [Mitchison DA, 1985]. Yet, the observation that M. tuberculosis strains lacking PZAse are resistant to both PZA and NCA [Konno K et al., 1967] appears to contradict the hypothesis that the interaction of NCA with chorismate synthase may represent a major contribution to the antimicrobial activity of NCA. Alternatively, there is the possibility that partial interruption of the shikimate pathway in mycobacteria ingested by phagocytes might be bypassed by the uptake of aromatic amino acids from the host. However, resistance against PZA or NCA may not necessarily be related to the absence or mutations of PZAse [Martin A et al., 2006; Singh P et al., 2006]. In total, the results presented in this work suggest taking the effect on chorismate production into account when further investigating the anti-TB activities of NCA and PZA.

The orientation of NCA in the complex with MtCS does not provide a model for the yet unknown orientation of the nicotinamide moiety of NADPH in interactions with bifunctional chorismate synthases. Crystal structures (1R52, 1R53) of the bifunctional chorismate synthase from Saccharomyces cerevisiae in unliganded form

[Quevillon-Figure 4-21: Superposition of mono- and bifunctional CS. Bifunctional ScCS (PDB-ID 1R52) is shown in purple, MtCS-NCA in green and MtCS-FMN in cyan; FMN is colored in red and NCA in yellow.

Cheruel S. et al., 2004] show a closely similar conformation as MtCS in the binding regions of FMN and NCA (Fig. 4-21). Constructing a meaningful structural model of possible NADPH binding to ScCS is not feasible due to complete disorder for 23% of the amino acids in 1R52 and 1R53. However, a superposition of NADPH onto NCA in the MtCS-NCA complex shows that binding of NADPH with a similar orientation of the nicotinamide moiety would require major intra-domain conformational changes of an extended part of the enzyme (Fig. 4-22a). Similar is true for ScCS. This possibility may be excluded considering the fact that crystal structures of other enzymes exhibiting interactions between FMN and NADPH ligands (e.g. 1HE4) show a different orientation, allowing the binding of the nicotinamide moiety with respect to the isoalloxazine ring of FMN in a similar orientation as EPSP (Fig. 4-22b). Based on spectroscopic and kinetic characterizations a similar arrangement has been proposed in earlier studies on bifunctional enzymes [Kitzing K et al., 2001] .

The binding mode of NCA proposes a way of stabilizing the unproductive active site conformation in MtCS and may provide a starting point for possible rational design of novel chorismate synthase inhibitors. This newly observed unproductive active site conformation is strongly dependent on the interaction of NCA with an invariant arginine that in turn prevents the correct enzymatic action of CS due to an interaction

A B

FMN NADPH

Figure 4-22: NADPH binding model in CS. A. Superposition of NADPH (grey) onto NCA (wheat) in the MtCS-NCA complex (green). Binding of NADPH would require major intra-domain conformational changes of an extended part of the enzyme. Clashing residues are shown red. B.

Interactions between FMN and NADPH ligands in the crystal structure of 1HE4 allow the binding of the nicotinamide moiety with respect to the isoalloxazine ring of FMN in a similar orientation as EPSP

NCA

NADPH

with an invariant aspartate. The mentioned arginine is likely involved in all key conformational changes of the enzymatic functioning and therefore may play a central role in ternary complex formation during the catalytic process. This special role of the invariant arginine has been validated by the application of MD simulations.

Therefore the structural and functional investigations presented on chorismate synthase in this work not only give detailed new insights into the biological function but also provide a basis for rational drug design studies on this pharmaceutically important enzyme.