Crystal structures of a novel family IV esterase in free and substrate-bound form.

Astrid Höppner,Manuel Ferrer,Karl‐Erich Jaeger,Cristina Coscolín,Stefanie Kobus,Sander H J Smits,Stephan Thies,Alexander Bollinger

doi:10.1111/febs.15680

Abstract

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).

Highlights

Lipolytic enzymes, that is, lipases and esterases, are among the most important biocatalysts in the field of biotechnology [1]
One different amino acid was identified at position 271, which is valine in PtEst1 and leucine in WP_073459097
The protein was produced as a Cterminal histidine-tag fusion protein with Escherichia coli LOBSTR [12] and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC), revealing a single band of about 40 Kilo dalton (kDa) molecular weight, as determined by SDS/PAGE (Fig. 1)

Summary

Introduction

That is, lipases and esterases, are among the most important biocatalysts in the field of biotechnology [1] These enzymes catalyze the reversible hydrolysis of carboxylic esters, producing a carboxylic acid and an alcohol, and they are classified by their function as carboxylic ester hydrolases (EC 3.1.1). The overall sequence identity among these enzymes is low, but they share specific conserved sequence motifs (e.g., the pentapeptide GXSXG, comprising the nucleophilic serine) owing to their common catalytic mechanism, which is composed of a nucleophile, acid, and base catalytic triad [3,4] Despite their high sequence diversity, most enzymes of the carboxylic ester hydrolase class show a canonical a/b-hydrolase fold, which is well explored by more than 1500 available protein structures according to the lipase engineering database [5]; only a few examples are known which show a bpropeller fold or an a-helix bundle structure [6]. The oxyanion hole is either build by the backbone amine of an amino acid next to a glycine (GX type), the last glycine of a triple glycine motif (GGGX type), or an amino acid with a large side chain (Y type) [5]

Methods

Results

Conclusion