Unveiling the Atomic-Level Determinants of Acylase-Ligand Complexes: An Experimental and Computational Study

The industrial production of higher-generation semisynthetic cephalosporins starts from 7-aminocephalosporanic acid (7-ACA), which is obtained by deacylation of the naturally occurring antibiotic cephalosporin C (CephC). The enzymatic process in which CephC is directly converted into 7-ACA by a cephalosporin C acylase has attracted industrial interest because of the prospects of simplifying the process and reducing costs. We recently enhanced the catalytic efficiency on CephC of a glutaryl acylase from Pseudomonas N176 (named VAC) by a protein engineering approach and solved the crystal structures of wild-type VAC and the H57βS-H70βS VAC double variant. In the present work, experimental measurements on several CephC derivatives and six VAC variants were carried out, and the binding of ligands into the VAC active site was investigated at an atomistic level by means of molecular docking and molecular dynamics simulations and analyzed on the basis of the molecular geometry of encounter complex formation and protein-ligand potential of mean force profiles. The observed significant correlation between the experimental data and estimated binding energies highlights the predictive power of our computational method to identify the ligand binding mode. The present experimental-computational study is well-suited both to provide deep insight into the reaction mechanism of cephalosporin C acylase and to improve the efficiency of the corresponding industrial process.