Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E.

Thomas Klaus,Daniel Wibberg,Andreas Albersmeier,Bettina Siebers,Christopher Bräsen,Kseniya S Zayulina,Markus Kaiser,Farnusch Kaschani,Tobias Busche,Jianbing Jiang,Sabrina Ninck,Ilya V Kublanov,Herman S Overkleeft,Alexander G Elcheninov,Jörn Kalinowski

doi:10.3389/fmicb.2021.734039

Abstract

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6–7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.

Highlights

Activity-based protein profiling (ABPP) is a powerful technique for the class-specific detection of active enzymes
Well established for eukaryotic (Niphakis and Cravatt, 2014; Morimoto and van der Hoorn, 2016) and bacterial (Keller et al, 2020) organisms, this methodology has only recently been applied in the Archaea for the first time and turned out to be likewise suitable for the identification of serine hydrolases under the extreme conditions many of the so far cultivated Archaea thrive in (Zweerink et al, 2017)
We suggest that EGIDFPOO_00532 (GH1) and EGIDFPOO_00674 (GH57) are involved in xylan degradation, contributing to the xylanolytic activity in Thermococcus sp. strain 2319x1E cells that was shown to be inducible upon growth on xylan, predominantly localized in cell membrane fractions (Figure 3) and targeted by the activity-based probe (ABP) JJB384 and JJB111 (Figure 5)

Summary

Introduction

Activity-based protein profiling (ABPP) is a powerful technique for the class-specific detection of active enzymes. ABPP has been proven as an elaborate tool for several mesophilic enzyme families, allowing to unravel the activity state of enzymes under different conditions without knowledge of their natural substrates or enzyme functions, and helps to deduce their function in certain cellular processes (Cravatt et al, 2008; Willems et al, 2014). This methodology can be applied to various types of biological samples and is even suitable for the in vivo study of enzyme activities under native conditions (Speers and Cravatt, 2004). JJB111 in turn showed activity toward a variety of βglycosidases, including β-xylosidases such as a bifunctional αL-arabinofuranosidase/β-D-xylosidase, β-galactosidases, and βglucuronidases (Chandrasekar et al, 2014; Husaini et al, 2018)

Methods

Results

Discussion

Conclusion