Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology

Abstract

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials.

Benchmarking of density functionals for the kinetics and thermodynamics of the hydrolysis of glycosidic bonds catalyzed by glycosidases

“…These reactions can happen between two carbohydrates or between a carbohydrate and a noncarbohydrate moiety (amino acids, e.g., in glycoproteins) . Due to the critical role of carbohydrates in human health as well as their possible use as fuel, there has been an increased interest in the study of glycosidases in recent years …”

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

“…Five glycosidases from the GH3 (PDB 3U48 and 5K6M), GH9 (PDB 1JS4 and 3X17), and GH116 (PDB 5BX5) families that shared the highest similarity to Bgl1D were selected for the alignment (Table S5). Blast results showed that Bgl1D shared approximately 16.57%, 27.49%, 24.56%, 20.78%, and 26.67% identities with the amino acids of 3U48, 5K6M [39], 1JS4 [40], 3X17 [41], and 5BX5 [42], respectively. A comparison of the amino acid of Bgl1D with these β-glucosidases demonstrated that the Asp44 of Bgl1D was similar to 3U48 (GH3 family) and 1JS4 (GH9 family) whose residue Asp was generally used as conserved catalytic nucleophile base or catalytic proton donor according to the information of β-glucosidase (EC 3.2.1.21) from the CAZy database (http://www.cazy.org) ( Figure 3) [43].…”

Identification and Immobilization of an Invertase With High Specific Activity and Sucrose Tolerance Ability of Gongronella sp. w5 for High Fructose Syrup Preparation

“…The alignment of the GspInv amino acid sequence with other characterized invertases revealed the GspInv contains conserved motifs of GH32 invertases (Supplementary Figure S1). Based on their alignments, residues D46, D173, and E251 of GspInv were identified as the nucleophile, transitionstate stabilizer, and general acid/base catalyst, respectively (Supplementary Figure S1; Pons et al, 2004;Ramírez-Escudero et al, 2016). Trollope et al (2015) reported that GH32 invertases can be divided into two groups, including that with lowand high-level fructooligosaccharide synthesis abilities based on the conserved motifs unique to each group.…”