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 H57ßS-H70ßS 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.

Strategic manipulation of an industrial biocatalyst--evolution of a cephalosporin C acylase.

Semi-synthetic cephalosporins are synthesized from the 7-amino cephalosporanic acid (7-ACA) nucleus produced from the antibiotic cephalosporin C (CephC). In recent years, a single-step enzymatic process in which CephC is directly converted into 7-ACA by a cephalosporin C acylase (CA) has attracted industrial interest because of the prospects of simplifying the process and reducing costs. CAs are members of the glutaryl acylase family that specifically use CephC as their substrate; however, known natural glutaryl acylases show very low activity on the antibiotic. We previously enhanced the catalytic efficiency on CephC of a glutaryl acylase from Pseudomonas N176 (named VAC) by a protein engineering approach, and solved the structures of the VAC, thus providing insight into the substrate binding and catalytic activity of CAs. However, the properties of such enzymes are not sufficient to encourage 7-ACA manufacturers to shift to single-step enzymatic conversion of CephC. Here, we combine structural knowledge, semi-rational design, computational approaches and evolution analysis to isolate VAC variants with altered substrate specificity (i.e. with a > 11,000-fold increase in specificity constant for CephC versus glutaryl-7-amino cephalosporanic acid, compared to wild-type) and with the highest kinetic efficiency so far obtained for a CA. Indeed, the H57ßS-H70ßS-L154ßY VAC variant shows the highest conversion of CephC into 7-ACA under conditions resembling those used at industrial level because of its high kinetic efficiency and the absence of substrate or product inhibition effects, and may be suitable for industrial application of the mono-step process for CephC conversion.