Influence of Cysteine 440 on the Active Site Properties of 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate Synthase in Mycobacterium tuberculosis (MtDAHPS)

[Image: see text] The shikimate pathway, which produces aromatic amino acids and key intermediates, is critical to the viability of the tuberculosis-causing pathogen Mycobacterium tuberculosis. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first committed step of this pathway and possesses regulatory functions. Its active site contains two cysteinyls: one (Cys(87)) bound to a metal ion, while the other (Cys(440)) is in proximity to the first but is located on a connecting loop. This arrangement seemingly appeared as a disulfide linkage. However, Cys(440) is not metal binding, and its positioning indicates that it could collapse the disulfide linkage. Hence, its potential role may be more than simply structural support of the active site fold. Using a multiscale computational approach, molecular dynamics (MD) simulations, and DFT-based calculations, the influence of Cys(440) on the active site properties has been investigated. MD simulations reveal an unusually long disulfide bond, more than 3.0 Å, whereas DFT calculations identified two stable active site conformers in the triplet and quintet spin states. Analysis of group spin density distribution identified antiferromagnetic coupling in each conformer, which suggests their relatively low potential energy and stable conformations. The conformer in the triplet spin state could favor enzyme reactivity due to its low HOMO–LUMO energy gap. In addition, reduction of the Cys(440) thiolate group results in collapse of the active site metal–ligand configuration with large exothermicity. Hence, Cys(440) could activate and inactivate the enzyme. For the first time, the study revealed the role of Cys(440) as being vital for the catalytic activity of the enzyme rather than solely for the structural stabilization of its active site. Thus, the findings may lead to a novel basis for antituberculosis drug design and development that would disrupt the contributions of the Cys(440).