Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus.

Yi Ding,Ying-Yi Huo,Xue-Wei Xu,Heng-Lin Cui,Jixi Li,Yan Gao,Yang Li,Xiao-Chen Yang,Laiyin Nie,Zhengyang Li

doi:10.3389/fmicb.2021.798194

Abstract

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.

Highlights

Catalyzing the hydrolysis and synthesis of lipids, lipolytic enzymes are essential enzymes in the scope of biological processes (Arpigny and Jaeger, 1999; Nardini and Dijkstra, 1999) and for industrial applications (Stergiou et al, 2013; Ferrer et al, 2016)
The catalytic reaction relies on the S-HD/E catalytic triad located in the catalytic domain
Results suggested that E53 exhibited good tolerance on several metal ions: activity was retained to more than 50% in all tested cations (Ba2+, Ca2+, Mg2+, Mn2+, Sr2+) and ethylenediaminetetraacetic acid (EDTA) did not show any inhibition to the enzymatic activity (Figure 2D)

Summary

Introduction

Catalyzing the hydrolysis and synthesis of lipids, lipolytic enzymes are essential enzymes in the scope of biological processes (Arpigny and Jaeger, 1999; Nardini and Dijkstra, 1999) and for industrial applications (Stergiou et al, 2013; Ferrer et al, 2016). Lipolytic enzymes can be classified into two groups, the lipases (EC 3.1.1.3) that degrade long-chain esters, and the esterases (EC 3.1.1.1) that degrade short-chain esters. According to their conserved sequence motifs and the biological properties, these enzymes could be classified into 18 families, family I-XVIII (Samoylova et al, 2018). The GXSAG motif contains the serine, one of the catalytic triad of the enzyme. According to the amino acid residue of X in the GXSAG motif, the family IV esterase can be further classified into GTSAG, GDSAG, and GCSAG subfamily (Jeon et al, 2011; Petrovskaya et al, 2016)

Methods

Results

Conclusion