A Catalytic Perspective on Erythromycin Hydrolysis by Erythromycin Esterase C: Combined CpHMD and QM/MM Metadynamics Study

Macrolide esterases, which are preponderant drug-inactivating enzymes present inside drug-resistant bacteria, pose a significant threat to the realm of macrolide antibiotics. They dissect the antibiotic's macrolactone ring structure by hydrolyzing the ester moiety, consequently diminishing the antimicrobial efficacy in treating bacterial infections. Motivated by the recently resolved crystallographic structure of an important member of the macrolide esterase - 'Erythromycin Esterase C (EreC)' - we have delved into the catalytic itineraries of the erythromycin hydrolysis utilizing QM/MM methods. To the best of our knowledge, this research work constitutes the first study accounting for two important aspects - EreC's conformational reliance on the local pH environment and intricate mechanistic details about erythromycin hydrolysis. We have demonstrated the pivotal role of Glu78 and Glu300 in modulating EreC^open/close conformational switching through rigorous constant pH molecular dynamics (CpHMD) simulations. This study also proclaims the subtle shift of the internal dipolar microenvironment inside the catalytic room, inducing a notable reduction in the p<i>K</i>_a value of His50. Thus, it can act as a putative general base which extracts a proton from the catalytic water that participates in nucleophilic addition toward the ester carbonyl center of the macrolactone erythromycin, confirming the foremost catalytic role of '<i>Glu47-His50 pair (Catalytic Dyad)</i>' postulated by previous structural studies. Employing QM/MM metadynamics, we have delineated the complete free energy landscape of the hydrolysis process, which unfolds through two successive steps. Moreover, the current study also spotlights the deprotonated state of Glu78 and conformational locking of Arg261 and His289, which is absolutely necessary for seamless hydrolysis of the erythromycin. Collectively, this work advances the mechanistic understanding of the metal-independent hydrolase EreC, highlighting pH-sensitive conformational switching and the nuanced orchestration of active-site residues in macrolide degradation.