Computational evidence of a new allosteric communication pathway between active sites and putative regulatory sites in the alanine racemase ofMycobacterium tuberculosis

Abstract

AbstractAlanine racemase, a popular drug target fromMycobacterium tuberculosis, catalyzes the biosynthesis of D-alanine, an essential component in bacterial cell walls. With the help of elastic network models of alanine racemase fromMycobacterium tuberculosis, we show that the mycobacterial enzyme fluctuates between two undiscovered states—a closed and an open state. A previous experimental screen identified several drug-like lead compounds against the mycobacterial alanine racemase, whose inhibitory mechanisms are not known. Docking simulations of the inhibitor leads onto the mycobacterial enzyme conformations obtained from the dynamics of the enzyme provide first clues to a putative regulatory role for two new pockets targeted by the leads. Further, our results implicate the movements of a short helix, behind the communication between the new pockets and the active site, indicating allosteric mechanisms for the inhibition. Based on our findings, we theorize that catalysis is feasible only in the open state. The putative regulatory pockets and the enzyme fluctuations are conserved across several alanine racemase homologs from diverse bacterial species, mostly pathogenic, pointing to a common regulatory mechanism important in drug discovery.Author summaryIn spite of the discovery of many inhibitors against the TB-causing pathogenMycobacterium tuberculosis, only a very few have reached the market as effective TB drugs. Most of the marketed TB drugs induce toxic side effects in patients, as they non-specifically target human cells in addition to pathogens. One such TB drug, D-cycloserine, targets pyridoxal phosphate moiety non-specifically regardless of whether it is present in the pathogen or the human host enzymes. D-cycloserine was developed to inactivate alanine racemase in TB causing pathogen. Alanine racemase is a bacterial enzyme essential in cell wall synthesis. Serious side effects caused by TB drugs like D-cycloserine, lead to patients’ non-compliance with treatment regimen, often causing fatal outcomes. Current drug discovery efforts focus on finding specific, non-toxic TB drugs. Through computational studies, we have identified new pockets on the mycobacterial alanine racemase and show that they can bind drug-like compounds. The location of these pockets away from the pyridoxal phosphate-containing active site, make them attractive target sites for novel, specific TB drugs. We demonstrate the presence of these pockets in alanine racemases from several pathogens and expect our findings to accelerate the discovery of non-toxic drugs against TB and other bacterial infections.