Reaction intensification for biocatalytic production of polyphenolic natural product di-C-ß-glucosides.

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-ß-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 ß-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-ß-glucoside synthesis from expedient substrates.

Enzymatic C4-Epimerization of UDP-Glucuronic Acid: Precisely Steered Rotation of a Transient 4-Keto Intermediate for an Inverted Reaction without Decarboxylation.

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 ß-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.

Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction.

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.

Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases.

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.

Solvent Engineering for Nonpolar Substrate Glycosylation Catalyzed by the UDP-Glucose-Dependent Glycosyltransferase UGT71E5: Intensification of the Synthesis of 15-Hydroxy Cinmethylin ß-d-Glucoside.

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 ß-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-ß-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 ß-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.

Enzymatic ß-elimination in natural product O- and C-glycoside deglycosylation.

Biological degradation of natural product glycosides involves, alongside hydrolysis, ß-elimination for glycosidic bond cleavage. Here, we discover an O-glycoside ß-eliminase (OGE) from Agrobacterium tumefaciens that converts the C3-oxidized O-ß-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 ß-elimination of 3-keto C-ß-D-glucosides. We show that CGE catalyzes ß-elimination of 3-keto O- and C-ß-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 ß-elimination of 3-keto O-ß-D-glucosides. C-Glycoside cleavage is a specialized feature of this active site which is elicited by substrate through finely tuned enzyme-aglycone interactions.

Mechanistic characterization of UDP-glucuronic acid 4-epimerase.

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 ß-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.

Stereo-electronic control of reaction selectivity in short-chain dehydrogenases: Decarboxylation, epimerization, and dehydration.

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, UDP-apiose/UDP-xylose synthase) and UDP-glucuronic acid-4-epimerase 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 Cα of the transient keto-hexose 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 ß-elimination of water from the keto-hexose species.

Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.

D-Apiose is a C-branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from Arabidopsis thaliana, molecular dynamics simulations expanded by QM/MM 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 coenzyme with retro-aldol/aldol chemistry for the sugar ring contraction. They further demonstrate that decarboxylation occurs only after the sugar ring opening and identify the thiol group of Cys100 in steering the sugar skeleton rearrangement by proton transfer to and from the 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.