Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius.

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+ß 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 α2ß2 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).

LolA and LolB from the plant-pathogen Xanthomonas campestris forms a stable heterodimeric complex in the absence of lipoprotein.

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.

The Gram-positive bacterium Romboutsia ilealis harbors a polysaccharide synthase that can produce (1,3;1,4)-ß-D-glucans.

(1,3;1,4)-ß-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)-ß-D-glucan biosynthesis in grasses. Little is known about the enzymes responsible for synthesizing (1,3;1,4)-ß-D-glucans outside the grasses. In the present study, we report the presence of (1,3;1,4)-ß-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)-ß-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)-ß-D-glucans are produced with structures similar to those of the (1,3;1,4)-ß-D-glucans of the lichen Cetraria islandica. Phylogenetic analysis reveals that putative (1,3;1,4)-ß-D-glucan synthase candidate genes in several other bacterial species support the finding of (1,3;1,4)-ß-D-glucans in these species.

Biochemical Characterization of Pyranose Oxidase from Streptomyces canus-Towards a Better Understanding of Pyranose Oxidase Homologues in Bacteria.

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.

Potential of unglycosylated horseradish peroxidase variants for enzyme prodrug cancer therapy.

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 plant-derived 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.

Ellagic acid and polyhydroxylated urolithins are potent catalytic inhibitors of human topoisomerase II: an in vitro study.

Ellagic acid (EA), a natural polyphenol abundant in fruits and common in our diet, is under intense investigation for its chemopreventive activity resulting from multiple effects. EA inhibits topoisomerase II, but the effects on the human enzyme of urolithins, its monolactone metabolites, are not known. Therefore, the action of several synthetic urolithins toward topoisomerases II was evaluated, showing that polyhydroxylated urolithins, EA, and EA-related compounds are potent inhibitors of the α and ß isoforms of human topoisomerase II at submicromolar concentrations. Competition tests demonstrate a dose-dependent relationship between ATP and the inhibition of the enzyme. Docking experiments show that the active compounds bind the ATP pocket of the human enzyme, thus supporting the hypothesis that EA and polyhydroxylated urolithins act as ATP-competitive inhibitors of human topoisomerase II.