Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel

• DOI: 10.1128/aem.01817-16
• Publication Date: 2016-09-10T01:51:22Z
• Authors: Xiaodan Yan; Jianjun Wang; Yu Sun; Junge Zhu; Sheng Wu
• Keywords: Esterase; Catalytic triad; Protein Engineering; Hydrolase

Promiscuous enzymes are generally considered to be starting points in the evolution of offspring with more specific or even novel catalytic activities, which is molecular basis producing new biological functions. Mhg, a typical α/β fold hydrolase, was previously reported have both γ-lactamase and perhydrolase activities. However, despite having high structural similarity sharing an identical triad extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any activity. Molecular docking sequence analysis suggested possible role for entry binding pocket blocking entrance tunnel, preventing ester compounds entering into pocket. By engineering tunnel only one two amino acid substitutions, we successfully obtained five variants Mhg. The exhibited very broad substrate acceptance, hydrolyzing classical p-nitrophenol esters but also various types chiral esters, widely used as drug intermediates. Site 233 at Mhg found play pivotal modulating three activities by adjusting size shape different substitutions site facilitating Remarkably, variant L233G mutation without Considering conservation differentiation, could key target future protein engineering. In addition, demonstrate that efficient strategy regulate capabilities.Promiscuous can act thus providing production study, identified critical residue (Leu233) promiscuous enzyme, We substitution smaller acids such Gly, Ala, Ser, Pro endowed Different facilitate These findings universal significance subset hydrolases, including Furthermore, evolve capabilities. Our study has important implications regulation promiscuity development methodologies.