The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans

Abstract

In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-␤(1,4)-glucanase or ␤(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme from Saccharophagus degradans (SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides. SdGluc5_26A displays lichenase activity toward ␤(1,3; 1,4)-glucans with a side cellobiohydrolase activity toward ␤(1, 4)-glucans. The three-dimensional structure of SdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region of SdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization of SdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity of SdGluc5_26A. Its deletion opens the enzyme cleft at the ؊3 subsite and turns the enzyme into an endo-␤(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 β-Xylosidases

“…The use of oligomerization to adapt to extreme environments or to change enzyme activity is not a distinguishing characteristic of GH39 family and other CAZy families also make use of this strategy such as GH1 (Zanphorlin et al, 2016), GH5 (Lafond et al, 2016), GH10 (Santos et al, 2014), and GH32 (Sainz-Polo et al, 2013). The unexpected occupation of the catalytic interface of XacXynB by a peptide segment from the N-terminal Histag revealed conserved interactions with sugars moieties based on structural superpositions with other GH39 β-xylosidases in complex with xylose and analogs.…”

Purification, characterization, immobilization and applications of an enzybiotic β-1,3–1,4-glucanase produced from halotolerant marine Halomonas meridiana ES021

“…SJ-10 cultures (Baek et al 2017 ; Tak et al 2019 ), and for both wild type and mutant β-1,3–1,4-glucanase purified from Bacillus subtilis MA139 (Pei et al 2015 ). Also, it was higher than that reported for recombinant β-1,3–1,4-glucanase purified from Saccharophagus degradans (30 °C) (Lafond et al 2016 ), and Bacillus velezensis ZJ20 (35 °C) (Xu et al 2016 ). The purified β-1,3–1,4-glucanase enzyme retained more than 80% of its activity in a temperature range from 30 to 70 °C similarly to Kim et al ( 2013 ), similar range was also reported for recombinant β-1,3–1,4-glucanase purified from Bacillus terquilensis CGX 5–2 while its wildtype could only maintain high activity in a temperature range from 35 to 55 °C (Niu et al 2017 ).…”

Phylogenomics reveals the evolutionary origins of lichenization in chlorophyte algae

“…To functionally test whether the LFA GH8 can degrade lichenan, we cloned orthologs from three species and successfully produced the recombinant GH8 from Asterochloris glomerata . Enzyme assays towards different polysaccharides confirmed a typical lichenase activity 45 for this enzyme with a significant cleavage of mixed linked β-1,3/β-1,4 glucans, while no cleavage was observed on cellulose nor chitosan substrates (Figure 4C ). The detailed chromatographic analysis of the major soluble products that accumulated over time upon enzyme action showed they do not correspond to β-1,4-linked cellooligosaccharides nor to β-1,3-linked laminari-oligosaccharides, thus suggesting that the degradation products belong to the mixed linked β-1,3/1,4 class.…”