C-Glycosyl flavonoids are structural isomers and metabolically stable analogues of the corresponding flavonoid O-glycosides. O → C rearrangement of O-glycoside substrates offers a promising route to C-glycosyl compounds, but its synthetic exploitation requires efficient (bio)catalytic methods. Here, we uncover activity of the C-glycosyltransferase from Fortunella crassifolia (FcCGT) for uridine 5'-diphosphate (UDP)-dependent deglycosylation of phlorizin (phloretin 2'-β-O-glucoside). This activity is undetectable in the reverse direction (O-glycosylation of phloretin) due to the enzyme's strong preference for C-glycosylation. Phlorizin deglycosylation releases UDP-glucose and phloretin, which are subsequently used by the enzyme in a C-glycosylation step to selectively form nothofagin (phloretin 3'-β-C-glucoside). O → C rearrangement of phlorizin in the presence of additional UDP-glucose affords phloretin 3',5'-di-β-C-glucoside as a single product in quantitative yield at a concentration of up to 25 mM (~15 g/L). Trilobatin (phloretin 4'-β-O-glucoside) is not accepted by FcCGT for O → C rearrangement, but can undergo C-glycosylation to yield a mixed O,C-diglucoside of phloretin. O → C rearrangement provides excellent atom economy for C-glucoside synthesis. Owing to the higher solubility of the O-glycosylated substrate compared to the free aglycone, C-glucoside synthesis via O → C rearrangement eliminates the need for solubilization strategies such as organic cosolvents or inclusion complexation.

UDP-glucuronic acid 4-epimerase (UGAepi) catalyzes the NAD+-dependent interconversion of UDP-glucuronic acid (UDP-GlcA) and UDP-galacturonic acid (UDP-GalA) through a mechanism involving C4-oxidation, 4-keto-intermediate rotation, and subsequent reduction. Here, the functional significance of the substrate's carboxylate group in the epimerization process was investigated using UDP-4-keto-pentose, an analogous intermediate that lacks a carboxylate moiety. Site-directed mutations were introduced into UGAepi from Bacillus cereus (BcUGAepi) to increase substrate binding pocket flexibility, enabling the variant enzymes to accommodate UDP-4-keto-pentose more efficiently than the wild-type does. Although these BcUGAepi variants partially maintained nonstereospecific C4-epimerization activity with UDP-GlcA, they demonstrated fully stereospecific reduction of UDP-4-keto-pentose to UDP-xylose. These findings highlight the critical role of the carboxylate moiety as an essential element for epimerization in BcUGAepi, and elucidate the structural determinants of substrate specificity in UGAepis.

The structural motif of hetero-di-C-glycosyl compound is prominent in plant polyphenol natural products and involves two different glycosyl residues (e.g., β-d-glucosyl, β-d-xylosyl) attached to carbons of the same phenolic ring. Polyphenol hetero-di-C-glycosides attract attention as specialized ingredients of herbal medicines and their tailored synthesis by enzymatic C-glycosylation is promising to overcome limitations of low natural availability and to expand molecular diversity to new-to-nature glycoside structures. However, installing these di-C-glycoside structures with synthetic precision and efficiency is challenging. Here we have characterized the syntheses of C-β-galactosyl-C-β-glucosyl and C-β-glucosyl-C-β-xylosyl structures on the phloroglucinol ring of the natural polyphenol phloretin, using kumquat (Fortunella crassifolia) C-glycosyltransferase (FcCGT). The FcCGT uses uridine 5'-diphosphate (UDP)-galactose (5 mU/mg) and UDP-xylose (0.3 U/mg) at lower activity than UDP-glucose (3 U/mg). The 3'-C-β-glucoside (nothofagin) is ~10-fold less reactive than non-glycosylated phloretin with all UDP-sugars, suggesting the practical order of hetero-di-C-glycosylation as C-galactosylation or C-xylosylation of phloretin followed by C-glucosylation of the resulting mono-C-glycoside. Each C-glycosylation performed in the presence of twofold excess of UDP-sugar proceeds to completion and appears to be effectively irreversible, as evidenced by the absence of glycosyl residue exchange at extended reaction times. Synthesis of C-β-glucosyl-C-β-xylosyl phloretin is shown at 10 mM concentration in quantitative conversion using cascade reaction of FcCGT and UDP-xylose synthase, allowing for in situ formation of UDP-xylose from the more expedient donor substrate UDP-glucuronic acid. The desired di-C-glycoside with Xyl or Gal was obtained as a single product of the synthesis and its structure was confirmed by NMR.

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions. Uridine diphosphate-dependent glycosyltransferases (UGTs) play a vital role in the biocatalytic glycosylation of phenolic compounds, however, UGT-catalyzed transformations remain not well-characterized. Here, the authors investigate new members of UGT72 and UGT84 families, revealing their specific reactivity and regio-selectivity on selected polyphenolic substrates.

AbstractUnderstanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ($${\Delta {{{{{\rm{H}}}}}}}^{{{\ddagger}} }$$ Δ H ‡ = 54 kJ mol−1), significant loss in entropy ($$-T{\Delta {{{{{\rm{S}}}}}}}^{{{\ddagger}} }$$ − T Δ S ‡ = 20 kJ mol−1; 298 K) and negative activation heat capacity ($${\Delta {{{{{\rm{C}}}}}}}_{{{{{{\rm{p}}}}}}}^{{{\ddagger}} }$$ Δ C p ‡ = −0.64 kJ mol−1 K−1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

The glycosylation of an alcohol group from a sugar nucleotide substrate involves proton release, so the reaction is favored thermodynamically at high pH. Here, we explored expansion of the alkaline pH range of sucrose synthase (SuSy; EC 2.4.1.13) to facilitate enzymatic glycosylation from uridine 5'-diphosphate (UDP)-glucose. The apparent equilibrium constant of the SuSy reaction (UDP-glucose + fructose ↔ sucrose + UDP) at 30 °C increases by ∼4 orders of magnitude as the pH is raised from 5.5 to 9.0. However, the SuSy in solution loses ≥80 % of its maximum productivity at pH ∼7 when alkaline reaction conditions (pH 9.0) are used. We therefore immobilized the SuSy on nanocellulose-based biocomposite carriers (∼48 U/g carrier; ≥ 50 % effectiveness) and reveal in the carrier-bound enzyme a substantial broadening of the pH-productivity profile to high pH, with up to 80 % of maximum capacity retained at pH 9.5. Using reaction by the immobilized SuSy with automated pH control at pH ∼9.0, we demonstrate near-complete conversion (≥ 96 %) of UDP-glucose and fructose (each 100 mM) into sucrose, as expected from the equilibrium constant (Keq = ∼7 × 102) under these conditions. Collectively, our results support the idea of glycosyltransferase-catalyzed synthetic glycosylation from sugar nucleotide donor driven by high pH; and they showcase a marked adaptation to high pH of the operational activity of the soybean SuSy by immobilization.

Biological degradation of natural product glycosides involves, alongside hydrolysis, beta-elimination for glycosidic bond cleavage. Here, we discover an O-glycoside beta-eliminase (OGE) from Agrobacterium tumefaciens that converts the C3-oxidized O-beta-d-glucoside of phloretin (a plant-derived flavonoid) into the aglycone and the 2-hydroxy-3-keto-glycal elimination product. While unrelated in sequence, OGE is structurally homologous to, and shows effectively the same Mn2+ active site as, the C-glycoside deglycosylating enzyme (CGE) from a human intestinal bacterium implicated in beta-elimination of 3-keto C-beta-D-glucosides. We show that CGE catalyzes beta-elimination of 3-keto O- and C-beta-D-glucosides while OGE is specific for the O-glycoside substrate. Substrate comparisons and mutagenesis for CGE uncover positioning of aglycone for protonic assistance by the enzyme as critically important for C-glycoside cleavage. Collectively, our study suggests convergent evolution of active site for beta-elimination of 3-keto O-beta-D-glucosides. C-Glycoside cleavage is a specialized feature of this active site which is elicited by substrate through finely tuned enzyme-aglycone interactions.

Biotechnology and BioengineeringVolume 120, Issue 6 p. 1451-1454 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 120, Number 6, June 2023 First published: 12 May 2023 https://doi.org/10.1002/bit.28139AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume120, Issue6June 2023Pages 1451-1454 RelatedInformation

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4-0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.

Sugar nucleotide-dependent glycosyltransferases are powerful catalysts of the glycosylation of natural products and xenobiotics. The low solubility of the aglycone substrate often limits the synthetic efficiency of the transformation catalyzed. Here, we explored different approaches of solvent engineering for reaction intensification of β-glycosylation of 15HCM (a C15-hydroxylated, plant detoxification metabolite of the herbicide cinmethylin) catalyzed by safflower UGT71E5 using UDP-glucose as the donor substrate. Use of a cosolvent (DMSO, ethanol, and acetonitrile; ≤50 vol %) or a water-immiscible solvent (n-dodecane, n-heptane, n-hexane, and 1-hexene) was ineffective due to enzyme activity and stability, both impaired ≥10-fold compared to a pure aqueous solvent. Complexation in 2-hydroxypropyl-β-cyclodextrin enabled dissolution of 50 mM 15HCM while retaining the UGT71E5 activity (∼0.32 U/mg) and stability. Using UDP-glucose recycling, 15HCM was converted completely, and 15HCM β-d-glucoside was isolated in 90% yield (∼150 mg). Collectively, this study highlights the requirement for a mild, enzyme-compatible strategy for aglycone solubility enhancement in glycosyltransferase catalysis applied to glycoside synthesis.

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone β-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.

UDP‐Glucuronsäure(UDP‐GlcA)‐4‐Epimerase repräsentiert eine wichtige Fragestellung in der Enzymkatalyse: die Balance zwischen konformativer Flexibilität und genauer Positionierung. Das Enzym koordiniert die C4‐Oxidation des Substrats durch NAD + mit der Rotation eines leicht decarboxylierbaren β‐Ketosäure‐Intermediats im aktiven Zentrum zur Ermöglichung der stereoinvertierenden Reduktion der Ketogruppe durch NADH. Wir zeigen hier die nur schwer erfassbare Rotationskoordinate des 4‐Ketointermediats. Distorsion des Zuckerrings in eine Boot‐Konformation erzeugt torsionale Mobilität in der Bindungstasche des Enzyms. Die Endpunkte der Rotation zeigen den 4‐Ketozucker in einer unverformten 4 C 1 ‐Sesselkonformation. Die äquatorial positionierte Carboxylatgruppe ist ungünstig für die 4‐Ketozucker‐Decarboxylierung. Varianten der Epimerase zeigen Decarboxylierung, wenn sie die Bindung mit der Carboxylatgruppe im entgegengesetzten Rotationsisomer des Substrats entfernen. R185A/D‐Substitutionen wandeln die Epimerase in UDP‐Xylose‐Synthasen um, welche UDP‐GlcA in stereospezifischen, konfigurationserhaltenden Reaktionen decarboxylieren.

Sugar nucleotide?modifying enzymes of the short-chain dehydrogenase/reductase type use transient oxidation?reduction by a tightly bound nicotinamide cofactor as a common strategy of catalysis to promote a diverse set of reactions, including decarboxylation, single- or double-site epimerization, and dehydration. Although the basic mechanistic principles have been worked out decades ago, the finely tuned control of reactivity and selectivity in several of these enzymes remains enigmatic. Recent evidence on uridine 5'-diphosphate (UDP)glucuronic acid decarboxylases (UDP-xylose synthase, UDPapiose/UDP-xylose synthase) and UDP-glucuronic acid-4epimerase suggests that stereo-electronic constraints established at the enzyme?s active site control the selectivity, and the timing of the catalytic reaction steps, in the conversion of the common substrate toward different products. The mechanistic idea of stereo-electronic control is extended to epimerases and dehydratases that deprotonate the Ca of the transient ketohexose intermediate. The human guanosine 5'-diphosphate (GDP)-mannose 4,6-dehydratase was recently shown to use a minimal catalytic machinery, exactly as predicted earlier from theoretical considerations, for the 0-elimination of water from the keto-hexose species.

UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a "swing and flip" rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4' atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.

UDP‐glucuronic acid (UDP‐GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4‐epimerases and decarboxylases releasing UDP‐galacturonic acid (UDP‐GalA) and UDP‐pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short‐chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme‐bound NAD+ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4‐epimerization of UDP‐GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP‐GlcA 4‐epimerase from Bacillus cereus functions as a homodimer and contains one NAD+/subunit (kcat = 0.25 ± 0.01 s−1). The epimerization of UDP‐GlcA proceeds via hydride transfer from and to the substrate’s C4 while retaining the enzyme‐bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The kcat for UDP‐GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP‐4‐keto‐hexose‐uronic acid intermediate whose formation is rate‐limiting overall, and is governed by a conformational step before hydride abstraction from UDP‐GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo‐electronic constraints in which decarboxylation of the labile β‐keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo‐electronic control may be a distinguishing feature of catalysis by SDR‐type epimerases and decarboxylases active on UDP‐GlcA.

Podophyllotoxin is probably the most prominent representative of lignan natural products. Deoxy-, epi-, and podophyllotoxin, which are all precursors to frequently used chemotherapeutic agents, were prepared by a stereodivergent biotransformation and a biocatalytic kinetic resolution of the corresponding dibenzylbutyrolactones with the same 2-oxoglutarate-dependent dioxygenase. The reaction can be conducted on 2 g scale, and the enzyme allows tailoring of the initial, "natural" structure and thus transforms various non-natural derivatives. Depending on the substitution pattern, the enzyme performs an oxidative C-C bond formation by C-H activation or hydroxylation at the benzylic position prone to ring closure.

The C-branched pentose sugar d-apiose is important for plant cell wall development. Its biosynthesis as urdine diphosphate-d-apiose (UDP-d-apiose) involves decarboxylation of the UDP-d-glucuronic acid precursor coupled with pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalysed in a single active site by UDP-d-apiose/UDP-d-xylose synthase (UAXS). Here we decipher the UAXS catalytic mechanism on the basis of crystal structures of the enzyme (which is from Arabidopsis thaliana), molecular dynamics simulations that are expanded by hybrid quantum mechanics/molecular mechanics calculations and mutational mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide co-enzyme with retroaldol/aldol chemistry for the sugar ring contraction. We further demonstrate that decarboxylation occurs only after the sugar ring opening and identify that the thiol group of Cys100 steers the sugar skeleton rearrangement by proton transfer to and from C3′. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis. The sugar d-apiose is important in plant cell wall polysaccharides. Here the authors elucidate the complex, multistep biosynthetic pathway for its production using enzyme crystal structures and computational analysis.

AbstractPodophyllotoxin (1) ist einer der bekanntesten Vertreter von natürlich vorkommenden Lignanen. Deoxy‐, epi‐ und Podophyllotoxin, welche alle Vorstufen für häufig verwendete chemotherapeutische Verbindungen darstellen, wurden mithilfe einer stereodivergenten Biotransformation und biokatalytischen kinetischen Racematspaltung der jeweiligen Dibenzylbutyrolactone durch dieselbe 2‐Oxoglutarat‐abhängige Dioxygenase (2‐ODD) hergestellt. Zudem konnte gezeigt werden, dass ein “Upscaling” der Reaktion auf 2 g möglich ist, sowie dass das 2‐ODD‐Enzym Modifikationen des “natürlichen” Substrates toleriert und somit verschiedenste nichtnatürliche Derivate umsetzen kann. Das Enzym führt je nach Substituentenmuster entweder eine oxidative C‐C‐Bindungsbildung durch C‐H‐Aktivierung oder Hydroxylierung an der für die Ringschließung zugänglichen benzylischen Position durch.