Here we describe a short study of how our in silico framework for enzyme engineering (eEF) can be applied to model and study the catalytic reaction coordinates of E-S reaction, identify hot spots in enzymes, generate more than a hundred thousand mutations, filter for the most appropriate E-S mutations and simulate E-S reaction to predict the kinetics, activation energies and rate limiting steps of the potential enzyme mutants.

The challenge undertaken was to engineer enzymes involved in the production of beta-lactam antibiotics. With annual sales of around $15 billion worth of penicillins and cephalosporins constituting close to 65 percent of the total antibiotics market [1], beta-Lactam antibiotics alone constitute most of the world's antibiotic sales (30 million kg/year out of a total 50 million kg/year produced worldwide [2]). There are battery of potential synthetic beta-lactam antibiotics produced using the chemical process, which in turn can be synthesized using the same enzyme that produce natural substrates like penicillins and cephalosporins. Moreover, enzyme-mediated conversion of beta-lactam antibiotics provides a novel direction for antibiotics industries and promotes a safer and cleaner environment [3] but this requires small or large transformations of these enzymes. The need to improve the present enzymatic reaction towards the natural substrates and attain activity for the synthetic substrates is set as foremost priority of enzyme engineering framework (eEF).

The mutagenesis carried out using eEF are based on the substrate binding path that is obtained from the kinetic pathway of the substrate/inhibitor diffusing from solvent to the bound state passing through different intermediate states. Rather than directly entering the binding pocket, the substrate/inhibitor appears to move on the surface of the enzyme in its transition states and then finally enter the catalytic site [4] (as shown in the Figure below).

This way the framework helps in identifying new hotspots that maybe missed out during a conventional enzyme engineering procedure. To identify hotspots in the enzyme we use different sampling techniques and the resultant trajectories are used to calculate inter-atomic weights for the residues in-and-around the active site. These weights are based on the physical contact and contact energies like Van der Waals forces, electrostatic, hydrogen bonds energies, pi-pi and cat-pi energies. Also, during the simulation process, we explore the path taken by the active substrates (high and low activity), inactive substrates (no activity) and substrates of interest to enter the active site of the enzyme. This way it helps in identifying modification sites even away from the binding site (identify new modification). This information is critical to engineer the enzyme, which will facilitate the substrate entry to the active site and the product dissociation forms the active site there by optimizing the whole enzymatic reaction.