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

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.

Glycerol degradation in the thermoacidophilic crenarchaeonSulfolobus acidocaldariusinvolves an unusual glycerol-3-phosphate dehydrogenase

“…After washing, 35 µl of LC-MS grade water was added and 10 µl of the bead solution was taken for SDS-PAGE analysis, Western blotting and immunodetection of HA-tagged proteins with anti-HA and horse radish peroxidase-coupled secondary antibodies using an iBright™ FL1500 (ThermoFisher). The remaining 25 µl bead suspension was used as input for on-bead digestion (Klaus et al, 2022).…”

“…After incubation, the tubes were placed once more on a magnetic stand and after 5 min the clear supernatant was again collected and combined with the recovered peptide mix, followed by the addition of trifluoroacetic acid (TFA; 2 % (w/v) final concentration) to the samples. Prior to LC-MS/MS analysis, peptides were desalted as described in (Klaus et al, 2022) with the only modification of not re-applying the flow-through to the C 18 StageTips again.…”

Chemoproteomic Approaches for Unraveling Prokaryotic Biology

“…strain 2319x1E, ABPP using cyclophellitol aziridinederived probes identified xylan-degrading enzymes with activities at high temperatures. [109] Collectively, these works highlight how ABPP has provided insight into the complex regulation of carbohydrate degradation by prokaryotes.…”

Anianabacter salinae gen. nov., sp. nov. ASV31T, a Facultative Alkaliphilic and Extremely Halotolerant Bacterium Isolated from Brine of a Millennial Continental Saltern

“…In particular, the CAZymes acting on glycosidic bonds have proven to be crucial for significant biotechnological advances within sectors that include bioenergy and bio-based (food/feed, materials and chemicals) industries. For example, the use in biotechnology of marine-fungi-derived CAZymes has been reported due to their ability to function at a high salinity, low water potential, high sodium ion concentration and under oligotrophic nutrient conditions, among other factors [62].…”