Rational Engineering of Multiple Active Sites in an Ester Hydrolase

Abstract

Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.

Effects of the Size, the Number, and the Spatial Arrangement of Reactive Patches on a Sphere on Diffusion-Limited Reaction Kinetics: A Comprehensive Study

“…Another example is a cell surface with multiple receptors for ligands [4]. Moreover, recently, an enzyme with multiple active sites was created by rational engineering [5], which implies that the number of examples will likely increase in the future. 2…”

Monte Carlo simulations using PELE to identify a protein–protein inhibitor binding site and pose

“…This basic algorithm has been applied to the study of multiple drug discovery [31][32][33] and protein engineering problems. 34,35 For the present work, the adaptive version of the algorithm was applied. Adaptive PELE is composed of three main steps: sampling, clustering, and spawning, which are run iteratively.…”

Application of the uridine auxotrophic host and synthetic nucleosides for a rapid selection of hydrolases from metagenomic libraries

“…The hunt for novel enzymes, including esterases, is usually based on one of the following strategies: (i) construction of metagenomic libraries, and subsequent functional screening (Martínez‐Martínez et al ., ; Ferrer et al ., ; Taupp et al ., ; Peña‐García et al ., ; Ferrer et al ., ; Steele et al ., ), (ii) (meta)genome mining based on the homology analysis, chemical synthesis of the target genes and evaluation of the catalytic properties of the recombinant proteins (Martínez‐Martínez et al ., ), (iii) directed evolution, including a high‐throughput scale (HTS) microfluidics screening of the combinatorial libraries of randomly generated enzyme variants (Colin et al ., ; Bunzel et al ., ), (iv) rational in silico design followed by an experimental verification of selected variants (Santiago et al ., ; Packer and Liu, ; Bornscheuer et al ., ; Fernández‐Álvaro et al ., ).…”