Structure-guided protein engineering increases enzymatic activities of the SGNH family esterases

Zhengyang Li; Long Li; Yingyi Huo; Zijun Chen; Yu Zhao; Jing Huang; Shuling Jian; Zhen Rong; Di Wu; Jianhua Gan; Xiaojian Hu; Jixi Li; Xue-Wei Xu

doi:10.1186/s13068-020-01742-8

Structure-guided protein engineering increases enzymatic activities of the SGNH family esterases

Biotechnol Biofuels. 2020 Jun 15:13:107. doi: 10.1186/s13068-020-01742-8. eCollection 2020.

Authors

Zhengyang Li^#¹, Long Li^#¹, Yingyi Huo², Zijun Chen¹, Yu Zhao¹, Jing Huang¹, Shuling Jian², Zhen Rong², Di Wu¹, Jianhua Gan¹, Xiaojian Hu¹, Jixi Li¹, Xue-Wei Xu²

Affiliations

¹ State Key Laboratory of Genetic Engineering, School of Life Sciences, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, 200438 China.
² Key Laboratory of Marine Ecosystem Dynamics, Ministry of Natural Resources, Ministry of Natural Resources & Second Institute of Oceanography, Hangzhou, 310012 China.

^# Contributed equally.

Abstract

Background: Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and therefore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. Many SGNH family esterases share high similarity in sequences. However, they have distinct enzymatic activities toward the same substrates. Due to a lack of structural information, the detailed catalytic mechanisms of these esterases remain barely investigated.

Results: In this study, we identified two SGNH family esterases, CrmE10 and AlinE4, from marine bacteria with significantly different preferences for pH, temperature, metal ion, and organic solvent tolerance despite high sequence similarity. The crystal structures of these two esterases, including wild type and mutants, were determined to high resolutions ranging from 1.18 Å to 2.24 Å. Both CrmE10 and AlinE4 were composed of five β-strands and nine α-helices, which formed one compact N-terminal α/β globular domain and one extended C-terminal domain. The aspartic residues (D178 in CrmE10/D162 in AlinE4) destabilized the conformations of the catalytic triad (Ser-Asp-His) in both esterases, and the metal ion Cd²⁺ might reduce enzymatic activity by blocking proton transfer or substrate binding. CrmE10 and AlinE4 showed distinctly different electrostatic surface potentials, despite the similar atomic architectures and a similar swap catalytic mechanism. When five negatively charged residues (Asp or Glu) were mutated to residue Lys, CrmE10 obtained elevated alkaline adaptability and significantly increased the enzymatic activity from 0 to 20% at pH 10.5. Also, CrmE10 mutants exhibited dramatic change for enzymatic properties when compared with the wide-type enzyme.

Conclusions: These findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

Keywords: Alkaline adaptability; Esterase; SGNH superfamily; Swapped structure.