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).

The Gram-negative bacterium Xanthomonas campestris is one of the most problematic phytopathogens, and especially the pathovar campestris (Xcc) that causes a devastating plant disease known as black rot and it is of considerable interest to understand the molecular mechanisms that enable virulence and pathogenicity. Gram-negative bacteria depend on lipoproteins (LPs) that serve many important functions including control of cell shape and integrity, biogenesis of the outer membrane (OM) and establishment of transport pathways across the periplasm. The LPs are localized to the OM where they are attached via a lipid anchor by a process known as the localization of lipoprotein (Lol) pathway. Once a lipid anchor has been synthesized on the nascent LP, the Lol pathway is initiated by a membrane-bound ABC transporter that extracts the lipid anchor of the LP from the IM. The ABC extractor presents the extracted LP to the transport protein LolA, which binds the anchor and thereby shields it from the hydrophilic periplasmic milieu. It is assumed that LolA then carries the LP across the periplasm to the OM. At the periplasmic face of the OM, the LP cargo is delivered to LolB, which completes the Lol pathway by inserting the LP anchor in the inner leaflet of the outer membrane. Earlier studies have shown that loss of Xcc LolA or LolB leads to decreased virulence and pathogenicity during plant infection, which motivates studies to better understand the Lol system in Xcc. In this study, we report the first experimental structure of a complex between LolA and LolB. The crystal structure reveals a stable LolA-LolB complex in the absence of LP. The structural integrity of the LP-free complex is safeguarded by specific protein-protein interactions that do not coincide with interactions predicted to participate in lipid binding. The results allow us to identify structural determinants that enable Xcc LolA to dock with LolB and initiate LP transfer.

(1,3;1,4)-β-D-Glucans are widely distributed in the cell walls of grasses (family Poaceae) and closely related families, as well as some other vascular plants. Additionally, they have been found in other organisms, including fungi, lichens, brown algae, charophycean green algae, and the bacterium Sinorhizobium meliloti. Only three members of the Cellulose Synthase-Like (CSL) genes in the families CSLF, CSLH, and CSLJ are implicated in (1,3;1,4)-β-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-β-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-β-D-glucans in the exopolysaccharides of the Gram-positive bacterium Romboutsia ilealis CRIBT. We also report that RiGT2 is the candidate gene of R. ilealis that encodes (1,3;1,4)-β-D-glucan synthase. RiGT2 has conserved glycosyltransferase family 2 (GT2) motifs, including D, D, D, QXXRW, and a C-terminal PilZ domain that resembles the C-terminal domain of bacteria cellulose synthase, BcsA. Using a direct gain-of-function approach, we insert RiGT2 into Saccharomyces cerevisiae, and (1,3;1,4)-β-D-glucans are produced with structures similar to those of the (1,3;1,4)-β-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-β-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-β-D-glucans in these species.

Pyranose oxidase (POx, glucose 2-oxidase; EC 1.1.3.10, pyranose:oxygen 2-oxidoreductase) is an FAD-dependent oxidoreductase and a member of the auxiliary activity (AA) enzymes (subfamily AA3_4) in the CAZy database. Despite the general interest in fungal POxs, only a few bacterial POxs have been studied so far. Here, we report the biochemical characterization of a POx from Streptomyces canus (ScPOx), the sequence of which is positioned in a separate, hitherto unexplored clade of the POx phylogenetic tree. Kinetic analyses revealed that ScPOx uses monosaccharide sugars (such as d-glucose, d-xylose, d-galactose) as its electron-donor substrates, albeit with low catalytic efficiencies. Interestingly, various C- and O-glycosides (such as puerarin) were oxidized by ScPOx as well. Some of these glycosides are characteristic substrates for the recently described FAD-dependent C-glycoside 3-oxidase from Microbacterium trichothecenolyticum. Here, we show that FAD-dependent C-glycoside 3-oxidases and pyranose oxidases are enzymes belonging to the same sequence space.

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.

Fighting cancer still relies on chemo- and radiation therapy, which is a trade-off between effective clearance of malignant cells and severe side effects on healthy tissue. Targeted cancer treatment on the other hand is a promising and refined strategy with less systemic interference. The enzyme horseradish peroxidase (HRP) exhibits cytotoxic effects on cancer cells in combination with indole-3-acetic acid (IAA). However, the plantderived enzyme is out of bounds for medical purposes due to its foreign glycosylation pattern and resulting rapid clearance and immunogenicity. In this study, we generated recombinant, unglycosylated HRP variants in Escherichia coli using random mutagenesis and investigated their biochemical properties and suitability for cancer treatment. The cytotoxicity of the HRP-IAA enzyme prodrug system was assessed in vitro with HCT-116 human colon, FaDu human nasopharyngeal squamous cell carcinoma and murine colon adenocarcinoma cells (MC38). Extensive cytotoxicity was shown in all three cancer cell lines: the cell viability of HCT-116 and MC38 cells treated with HRP-IAA was below 1% after 24 h incubation and the surviving fraction of FaDu cells was <= 10% after 72 h. However, no cytotoxic effect was observed upon in vivo intratumoral application of HRP-IAA on a MC38 tumor model in C57BL/6J mice. However, we expect that targeting of HRP to the tumor by conjugation to specific antibodies or antibody fragments will reduce HRP clearance and thereby enhance therapy efficacy.

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.

Background: Propionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess.

Results: Adaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR-Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids.

Conclusion: Our study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.