Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2β2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min−1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min−1).

Dyes are coloring agents widely used in the textile and leather industries. Synthetic dyes are the major contributor to global wastewater exhibiting toxic effects on humans, aquatic organisms, and the environment. The conventional decolorization treatments are less effective due to high dye concentration in wastewater, complex chemical structure, and poor biodegradability. Several strategies like physical, chemical, and biological were reported earlier for dye decolorization. However, the adsorption technique has been extensively considered from economic and ecological standpoints. Biochar, a carbonaceous material generated by pyrolyzing waste biomass has been discussed here to tackle the dye-containing wastewater. As an adsorbent, biochar has gained considerable attention owing to its underlying advantages like larger surface area, high porosity, low production cost, varied surface functional groups, easy surface modifications, exceptional hydrophobicity, and aromaticity. In this chapter, we have discussed approaches for the production, and characterization of the biochar. Furthermore, we have also considered the effect of different physicochemical properties, sorption kinetics, isotherm models, and other key factors affecting the sorption mechanism. In this perspective, an effort has been made in this chapter to explore the probability and practicability of biochar as a sorbent for removal of the textile dyes.

Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan.

The impact of various β-glucans on the gut microbiome and immune system of vertebrates is becoming increasingly recognized. Besides the fundamental interest in understanding how β-glucans support human and animal health, enzymes that metabolize β-glucans are of interest for hemicellulose bioprocessing. Our earlier metagenomic analysis of the moose rumen microbiome identified a gene coding for a bacterial enzyme with a possible role in β-glucan metabolization. Here, we report that the enzyme, mrbExg5, has exo-β-1,3-glucanase activity on β-1,3-linked glucooligosaccharides and laminarin, but not on β-1,6- or β-1,4-glycosidic bonds. Longer oligosaccharides are good substrates, while shorter substrates are readily transglycosylated into longer products. The enzyme belongs to glycoside hydrolase subfamily GH5_44, which is a close phylogenetic neighbor of the subfamily GH5_9 exo-β-1,3-glucanases of the yeasts Saccharomyces cerevisiae and Candida albicans. The crystal structure shows that unlike the eukaryotic relatives, mrbExg5 is a functional homodimer with a binding region characterized by: (i) subsite +1 can accommodate a branched sugar on the β-1,3-glucan backbone; (ii) subsite +2 is restricted to exclude backbone substituents; and (iii) a fourth subsite (+3) formed by a unique loop. mrbExg5 is the first GH5_44 enzyme to be structurally characterized, and the first bacterial GH5 with exo-β-1,3-glucanase activity.

The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-beta-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-beta-1,4-mannosidase [EC 3.2.1.25] that is highly specific for beta-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 angstrom resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family beta-galactosidases and the GH164-family exo-beta-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 beta-galactosidase-type homotrimeric structure.

Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycanbiosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with "half' of the sixtransmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this "half tunnel" would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.

Commensal and pathogenic bacteria have evolved efficient enzymatic pathways to feed on host carbohydrates, including protein-linked glycans. Most proteins of the human innate and adaptive immune system are glycoproteins where the glycan is critical for structural and functional integrity. Besides enabling nutrition, the degradation of host N-glycans serves as a means for bacteria to modulate the host's immune system by for instance removing N-glycans on immunoglobulin G. The commensal bacterium Cutibacterium acnes is a gram-positive natural bacterial species of the human skin microbiota. Under certain circumstances, C. acnes can cause pathogenic conditions, acne vulgaris, which typically affects 80% of adolescents, and can become critical for immunosuppressed transplant patients. Others have shown that C. acnes can degrade certain host O-glycans, however, no degradation pathway for host N-glycans has been proposed. To investigate this, we scanned the C. acnes genome and were able to identify a set of gene candidates consistent with a cytoplasmic N-glycan-degradation pathway of the canonical eukaryotic N-glycan core. We also found additional gene sequences containing secretion signals that are possible candidates for initial trimming on the extracellular side. Furthermore, one of the identified gene products of the cytoplasmic pathway, AEE72695, was produced and characterized, and found to be a functional, dimeric exo-β-1,4-mannosidase with activity on the β-1,4 glycosidic bond between the second N-acetylglucosamine and the first mannose residue in the canonical eukaryotic N-glycan core. These findings corroborate our model of the cytoplasmic part of a C. acnes N-glycan degradation pathway.

The impact of various β-glucans on the gut microbiome and immune system of vertebrates is becoming increasingly recognized. Besides the fundamental interest in understanding how β-glucans support human and animal health, enzymes that metabolize β-glucans are of interest for hemicellulose bioprocessing. Our earlier metagenomic analysis of the moose rumen microbiome identified a gene coding for a bacterial enzyme with a possible role in β-glucan metabolization. Here, we report that the enzyme,mrbExg5, has exo-β-1,3-glucanase activity on β-1,3-linked glucooligosaccharides and laminarin, but not on β-1,6-or β-1,4-glycosidic bonds. Longer oligosaccharides are good substrates, while shorter substrates arereadily transglycosylated into longer products. The enzyme belongs to glycoside hydrolase subfamily GH5_44, which is a close phylogenetic neighbor of the subfamily GH5_9 exo-β-1,3-glucanases of Saccharomyces cerevisiaeand Candida albicans. The crystal structure shows that unlike the eukaryotic relatives, mrbExg5 is a functional homodimer with a binding region characterized by: (i) subsite +1 can accommodate a branched sugar on the β-1,3-glucan backbone; (ii) subsite +2 is restricted to exclude backbone substituents; and (iii) a fourth subsite (+3) formed by a unique loop. mrbExg5 is the first GH5_44 enzyme to be structurally characterized, and the first bacterial GH5 with exo-β-1,3-glucanase activity.