Combined Theoretical and Experimental Study to Unravel the Differences in Promiscuous Amidase Activity of Two Nonhomologous Enzymes

Abstract

Convergent evolution has resulted in nonhomologous enzymes that contain similar active sites that catalyze the same primary and secondary reactions. Comparing how these enzymes achieve their reaction promiscuity can yield valuable insights to develop functions from the optimization of latent activities. In this work, we have focused on the promiscuous amidase activity in the esterase from Bacillus subtilis (Bs2) and compared with the same activity in the promiscuous lipase B from Candida antarctica (CALB). The study, combining multiscale quantum mechanics/molecular mechanics (QM/MM) simulations, deep machine learning approaches, and experimental characterization of Bs2 kinetics, confirms the amidase activity of Bs2 and CALB. The computational results indicate that both enzymes offer a slightly different reaction environment reflected by electrostatic effects within the active site, thus resulting in a different reaction mechanism during the acylation step. A convolutional neural network (CNN) has been used to understand the conserved amino acids among the evolved protein family and suggest that Bs2 provides a more robust protein scaffold to perform future mutagenesis studies. Results derived from this work will help reveal the origin of enzyme promiscuity, which will find applications in enzyme (re)design, particularly in creating a highly active amidase.

Computational design of an amidase by combining the best electrostatic features of two promiscuous hydrolases

“…44 Due to the lack of crystallized structure for this specific variant, the framework for the required model, a natural variant of the protein (strain 168) was prepared based on a PNB esterase from a different organism (Bs2; PDB ID: 1QE3). 30 Details can be found in previous publications 25 and the Supporting Information. Based on the analysis of the previously computed FES of the amidase reaction catalyzed by wild-type CALB 24 and Bs2 25 with the N-(4-nitrophenyl)-butyramide as substrate, the structural alignment of the two proteins was subsequently done within the TS1 structures localized at QM/MM level in both systems.…”

“… 44 Due to the lack of a crystallized structure for this specific variant, the framework for the required model, a natural variant of the protein (strain 168) was prepared based on a PNB esterase from a different organism (Bs2; PDB ID: ). 30 Details can be found in previous publications 25 and the ESI. † Based on the analysis of the previously computed FES of the amidase reaction catalyzed by wild-type CALB 24 and Bs2 (ref.…”

“…† Based on the analysis of the previously computed FES of the amidase reaction catalyzed by wild-type CALB 24 and Bs2 (ref. 25 ) with N -(4-nitrophenyl)-butyramide as the substrate, the structural alignment of the two proteins was subsequently done within the TS1 structures localized at the QM/MM level in both systems. The QM sub-set of atoms was described with the M06-2X hybrid functional 45 and the solvent water molecules and the protein treated with the TIP3P 46 and the OPLS-AA 47 force field, respectively, as implemented in the fDynamo library.…”

“…31 We envisaged that creating mutations to optimize the preorganization of the protein environment will result in a variant that exhibits improved activity for the desired reaction. 32 Based on our recent 7 QM/MM studies of different enzymatic reactions, we have quantified and shown how the reactivity of different proteins can be rationalized from their electrostatic properties, 24,25,[33][34][35][36] as the pioneering works reported by Warshel and co-workers. [37][38][39] The computed changes of the electrostatic potential or the electric field exerted by the studied proteins on key atoms of the substrates reflect that there is a small reorganization of these entities when evolving from the reactant state (RS) to the TS at the lowest energy cost.…”

“…24,25 QM/MM studies of the amidase reaction catalyzed by the wild-type Bs2 and CALB enzymes were previously conducted. 24,25 We expect that favorable features of each enzyme could be isolated and combined to create redesign enzyme with improved catalytic activity for the secondary amidase reaction. In the present paper, based on our knowledge derived from previous comparative studies, and by applying the concept of electrostatic pre-organization, 24,[33][34][35][36][40][41][42][43] a variant with improved activity for the designated amidase reaction was generated.…”