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

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

doi:10.1186/s13068-020-01742-8

Zhengyang Li, Yingyi Huo + Show 11 more

Open Access

https://doi.org/10.1186/s13068-020-01742-8

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Abstract

BackgroundEsterases 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.ResultsIn 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.ConclusionsThese findings offer a perspective for understanding the catalytic mechanism of different esterases and might facilitate the industrial biocatalytic applications.

Highlights

Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and there‐ fore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries
The results showed that both CrmE10 and AlinE4 belong to the SGNH-hydrolase superfamily and the bacterial lipolytic enzyme GDSL family (Additional file 1: Fig. S1)
The presence of strictly conserved Ser, Gly, Asn, and His in blocks I, II, III, and V, respectively, confirmed that these two enzymes were new members of the SGNH-hydrolase family [4] (Additional file 1: Fig. S2)

Summary

Introduction

Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides, respectively, and there‐ fore play essential roles in the synthesis and decomposition of ester bonds in the pharmaceutical and food industries. The SGNH-hydrolase family, a superfamily that includes the bacterial lipolytic enzyme GDSL family [1], consists of enzymes possessing four strictly conserved residues, Ser, Gly, Asn, and His, in four conserved blocks, I, II, III, and V, respectively [2, 3]. Among these four residues, Ser and His serve as catalytic residues, and Ser, Gly, and Asn serve as oxyanion hole residues [2, 3]. Benefiting from the development of high-throughput sequencing technology, numerous SGNH hydrolases are found in the genomes of microorganisms, but the function and catalytic mechanism of microbial SGNH hydrolases remain unclear and need to be further explored

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