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KL55, KL74, and KL85 capsular polysaccharide (CPS) biosynthesis loci in Acinetobacter baumannii BAL_204, BAL_309, and LUH5543 genomes, respectively, are related and each contains genes for l-Rhap and d-GlcpA synthesis. The CPSs were isolated and studied by sugar analysis, Smith degradation, and 1 H and 13 C NMR spectroscopy. The K55 and K74 CPSs are built up of branched octasaccharide repeats (K units) containing one residue each of d-GlcpA and d-GlcpNAc and six residues of l-Rhap. The K55 unit differs from the K74 unit in the linkage between D-GlcpA and an l-Rhap residue in the K unit (1 → 3 versus 1 → 2) and linkage between K units. However, most K units in the isolated K74 CPS were modified by β-elimination of a side-chain α-l-Rhap-(1 → 3)-α-l-Rhap disaccharide from position 4 of GlcA to give 4-deoxy-l-threo-hex-4-enuronic acid (1:~3 ratio of intact and modified units). The K85 CPS has a branched heptasaccharide K unit similar to the K74 unit but with one fewer α-l-Rhap residue in the side chain. In contrast to previous findings on A. baumannii CPSs, each K locus includes fewer glycosyltransferase (Gtr) genes than the number required to form all linkages in the K units. Hence, one Gtr appears to be multifunctional catalysing formation of two 1 → 2 and one 1 → 3 linkages between the l-Rha residues., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-β-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus ), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus ), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus ), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter-dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-β-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Zorzoli et al.)

Isorhamnetin-3- O -rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli . The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The V max and K m for isorhamnetin were 0.646 U/mg and 181 μM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3- O -rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3- O -rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3- O -rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.

Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP-β-l-rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP-β-l-rhamnose. The mutant H357Q exhibited activity with both UDP-β-l-rhamnose and UDP-glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor-His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The uridine diphosphate glycosyltransferase (UGT) plays the central role in glycosylation of small molecules by transferring sugars to various acceptors including bioactive natural products in plants. UGT89C1 from Arabidopsis thaliana is a novel UGT, a rhamnosyltransferase, specifically recognizes UDP-l-rhamnose as donor. To provide an insight into the sugar specificity for UDP-l-rhamnose and interactions between UGT89C1 and its substrates, the UGT89C1 was expressed in Escherichia coli and purified toward biochemical and structural studies. Enzyme activity assay was performed, and the recombinant UGT89C1 recognized UDP-l-rhamnose and rhamnosylated kaempferol. Crystals of AtUGT89C1 were obtained, they diffracted to 2.7 Å resolution and belonged to space group I4 1 . AtUGT89C1 was also co-crystallized with UDP. Interestingly, two crystal forms were obtained in the same crystallization condition, including the previous I4 1 crystal form, and the new crystal form that diffracted to 3.0 Å resolution and belonged to space group P2 1 ., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat.