Molecular dynamics studies of alanine racemase: a structural model for drug design

Alanine racemase (AlaR) is a bacterial enzyme that catalyzes the interconversion of L- and D-alanine, which is an essential constituent of the peptidoglycan layer of the bacterial cell wall and requires pyridoxal 5'-phosphate (PLP) as a cofactor. The enzyme is universal to bacteria, including mycobacteria, making it an attractive target for drug design. To investigate the effects of flexibility on the binding modes of the substrate and an inhibitor and to analyze how the active site is affected by the presence of the substrate versus inhibitor, a molecular dynamics simulation on the full AlaR dimer from Bacillus stearothermophilus (pdb code: 1SFT) with a D-alanine molecule in one active site and the noncovalent inhibitor, propionate, in the second site has been carried out. Within the time scale of the simulation, we show that the active site becomes more stabilized in the presence of substrate versus inhibitor. The results of this simulation are in agreement with the proposed mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by acting cooperatively with Tyr 265' and Lys 39. A structural water molecule in contact with both substrate and inhibitor (i.e., in both active sites) and bridging residues in both active sites was identified. It shows a remarkably low mobility and does not exchange with bulk water. This water molecule can be taken into account for the design of specific AlaR inhibitors by either utilizing it as a bridging group or displacing it with an inhibitor atom. The results presented here provide insights into the dynamics of the alanine racemase in the presence of substrate/inhibitor, which will be used for the rational design of novel inhibitors.