ABSTRACT
Flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions. Here, based on study of enzymes from the phytopathogen Agrobacterium tumefaciens, we reveal a conserved Mn2+ metallocenter active site in both lyases and identify the structural requirements for specific catalysis to elimination of 3-keto-glucosides and water addition to the resulting 2-hydroxy-3-keto-glycal product, yielding 3-keto-glucose. Extending our search of GUL-encoded putative lyases to the human gut commensal Bacteroides thetaiotaomicron, we discover a Ca2+ metallocenter active site in a putative glycoside hydrolase-like protein and demonstrate its catalytic function in the eliminative cleavage of 3-keto-glucosides of opposite (alpha) anomeric configuration as preferred by the A. tumefaciens enzyme (beta). Findings identify a basic set of GUL-encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in bacteria.
ABSTRACT
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.
ABSTRACT
Understanding 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 ( Δ H = 54 kJ mol-1), significant loss in entropy ( - T Δ S = 20 kJ mol-1; 298 K) and negative activation heat capacity ( 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.
Subject(s)
Catalytic Domain , Thermodynamics , Kinetics , Protein Conformation , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/metabolism , Carbohydrate Epimerases/genetics , Biocatalysis , Catalysis , Models, MolecularABSTRACT
Here we report the efficient synthetic access to 13C/15N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75â nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63â nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75â nt E. coli tRNAGly CCmnm5U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids.
Subject(s)
Escherichia coli , Isotope Labeling , Nitrogen Isotopes , Organophosphorus Compounds , Pseudouridine , Pseudouridine/chemistry , Organophosphorus Compounds/chemistry , Nitrogen Isotopes/chemistry , Isotope Labeling/methods , RNA/chemistry , Carbon Isotopes/chemistry , Magnetic Resonance Spectroscopy/methodsABSTRACT
Linear d-glucans are natural polysaccharides of simple chemical structure. They are comprised of d-glucosyl units linked by a single type of glycosidic bond. Noncovalent interactions within, and between, the d-glucan chains give rise to a broad variety of macromolecular nanostructures that can assemble into crystalline-organized materials of tunable morphology. Structure design and functionalization of d-glucans for diverse material applications largely relies on top-down processing and chemical derivatization of naturally derived starting materials. The top-down approach encounters critical limitations in efficiency, selectivity, and flexibility. Bottom-up approaches of d-glucan synthesis offer different, and often more precise, ways of polymer structure control and provide means of functional diversification widely inaccessible to top-down routes of polysaccharide material processing. Here the natural and engineered enzymes (glycosyltransferases, glycoside hydrolases and phosphorylases, glycosynthases) for d-glucan polymerization are described and the use of applied biocatalysis for the bottom-up assembly of specific d-glucan structures is shown. Advanced material applications of the resulting polymeric products are further shown and their important role in the development of sustainable macromolecular materials in a bio-based circular economy is discussed.
Subject(s)
Biocatalysis , Glucans , Glucans/chemistry , Glycosyltransferases/metabolism , Glycoside Hydrolases/metabolism , PolymerizationABSTRACT
Enzymatic activity depends on and can therefore be regulated by temperature. Selective modulation of the activity of different enzymes in one reaction pot would require temperature control local to each type of enzyme. It has been suggested previously that immobilization of enzyme on magnetic nanoparticles and exposing them to alternating magnetic field can enhance the reaction rate. This enhancement has been explained as being mediated by temperature increase caused by dissipation of the absorbed field energy in the form of heat. However, the possibility of spatially limiting this temperature increase on the microscale has been questioned. Here, it is investigated whether an activity enhancement of the enzyme sucrose phosphorylase immobilized on magnetic beads can be achieved, how this effect is related to the increase in temperature, and whether temperature differences within one reaction pot could be generated in this way. It is found that alternating magnetic field stimulation leads to increased enzymatic activity fully attributable to the increase of bulk temperature. Both theoretical analysis and experimental data indicate that no local heating near the particle surface takes place. It is further concluded that relevant increase of surface temperature can be obtained only with macroscopic, millimeter-sized, magnetic particles.
Subject(s)
Enzyme Activation , Temperature , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Magnetic Fields , Glucosyltransferases/metabolismABSTRACT
The cellulosome is a megadalton-size protein complex that functions as a biological nanomachine of cellulosic fiber degradation. We show that the cellulosome behaves as a Brownian ratchet that rectifies protein motions on the cellulose surface into a propulsion mechanism by coupling to the hydrolysis of cellulose chains. Movement on cellulose fibrils is unidirectional and results from "macromolecular crawl" composed of dynamic switches between elongated and compact spatial arrangements of enzyme subunits. Deletion of the main exocellulase Cel48S eliminates conformational bias for aligning the subunits to the long fibril axis, which we reveal as crucial for optimum coupling between directional movement and substrate degradation. Implications of the cellulosome acting as a mechanochemical motor suggest a distinct mechanism of enzymatic machinery in the deconstruction of cellulose assemblies.
ABSTRACT
l-Lactate oxidase has important applications in biosensing and finds increased use in biocatalysis. The enzyme has been characterized well, yet its immobilization has not been explored in depth. Here, we studied immobilization of Aerococcus viridansl-lactate oxidase on porous carriers of variable matrix material (polymethacrylate, polyurethane, agarose) and surface functional group (amine, Ni2+-loaded nitrilotriacetic acid (NiNTA), epoxide). Carrier activity (Ac) and immobilized enzyme effectiveness (ɳ) were evaluated in dependence of protein loading. Results show that efficient immobilization (Ac: up to 1450â¯U/g carrier; ɳ: up to 65%) requires a hydrophilic carrier (agarose) equipped with amine groups. The value of ɳ declines sharply as Ac increases, probably due to transition into diffusional regime. Untagged l-lactate oxidase binds to NiNTA carrier similarly as N-terminally His-tagged enzyme. Lixiviation studies reveal quasi-irreversible enzyme adsorption on NiNTA carrier while partial release of activity (≤ 25%) is shown from amine carrier. The desorbed enzyme exhibits the same specific activity as the original l-lactate oxidase. Collectively, our study identifies basic requirements of l-lactate oxidase immobilization on solid carrier and highlights the role of ionic interactions in enzyme-surface adsorption.
Subject(s)
Aerococcus , Aerococcus/metabolism , Sepharose , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Enzymes, Immobilized/metabolism , AminesABSTRACT
The inherent complexity of coupled biocatalytic reactions presents a major challenge for process development with one-pot multienzyme cascade transformations. Kinetic models are powerful engineering tools to guide the optimization of cascade reactions towards a performance suitable for scale up to an actual production. Here, we report kinetic model-based window of operation analysis for cellobiose production (≥100 g/L) from sucrose and glucose by indirect transglycosylation via glucose 1-phosphate as intermediate. The two-step cascade transformation is catalyzed by sucrose and cellobiose phosphorylase in the presence of substoichiometric amounts of phosphate (≤27 mol% of substrate). Kinetic modeling was instrumental to uncover the hidden effect of bulk microviscosity due to high sugar concentrations on decreasing the rate of cellobiose phosphorylase specifically. The mechanistic-empirical hybrid model thus developed gives a comprehensive description of the cascade reaction at industrially relevant substrate conditions. Model simulations serve to unravel opposed relationships between efficient utilization of the enzymes and maximized concentration (or yield) of the product within a given process time, in dependence of the initial concentrations of substrate and phosphate used. Optimum balance of these competing key metrics of process performance is suggested from the model-calculated window of operation and is verified experimentally. The evidence shown highlights the important use of kinetic modeling for the characterization and optimization of cascade reactions in ways that appear to be inaccessible to purely data-driven approaches.
Subject(s)
Cellobiose , Phosphorylases , Cellobiose/chemistry , Glucosyltransferases/chemistry , Glucose , Sucrose , PhosphatesABSTRACT
One-pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α-d-glucose 1-phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step-by-step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial-rate models of the two-substrate reactions of sucrose phosphorylase (sucrose + phosphate â G1P + fructose) and cellobiose phosphorylase (G1P + glucose â cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two-site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.
Subject(s)
Cellobiose , Glucosyltransferases , Glucosyltransferases/metabolism , Phosphorylases/metabolism , Glucose , Disaccharides , Sucrose , Kinetics , Phosphates , Substrate SpecificityABSTRACT
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.
Subject(s)
Cardiac Glycosides , Glycosides , Humans , Glycosides/chemistry , Flavonoids/metabolism , Glucosides/metabolism , Intestines/microbiology , Substrate SpecificityABSTRACT
BACKGROUND: In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3'-sialyllactose (3SL). RESULTS: Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5'-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in â¼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of â¼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. CONCLUSIONS: Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.
Subject(s)
Escherichia coli , N-Acylneuraminate Cytidylyltransferase , Humans , N-Acylneuraminate Cytidylyltransferase/genetics , N-Acylneuraminate Cytidylyltransferase/metabolism , Escherichia coli/metabolism , Oligosaccharides/metabolism , Bioengineering , Sialyltransferases/genetics , Sialyltransferases/metabolism , CatalysisABSTRACT
Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near - but not in - the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model ß-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.
Subject(s)
Carbohydrates , Glycoside Hydrolases , Glycoside Hydrolases/metabolism , Proof of Concept Study , LigandsABSTRACT
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 (â¼0.32 U/mg) and stability. Using UDP-glucose recycling, 15HCM was converted completely, and 15HCM ß-d-glucoside was isolated in 90% yield (â¼150 mg). Collectively, this study highlights the requirement for a mild, enzyme-compatible strategy for aglycone solubility enhancement in glycosyltransferase catalysis applied to glycoside synthesis.
Subject(s)
Glycosyltransferases , Uridine Diphosphate Glucose , Glycosylation , Glycosyltransferases/genetics , Solvents , Glucosides , Water , Catalysis , GlucoseABSTRACT
Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.
Subject(s)
Fluorodeoxyglucose F18 , Trehalose , Humans , Cellobiose , Staphylococcus aureus , Positron-Emission Tomography/methods , BacteriaABSTRACT
Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach, that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG), the most common tracer used in clinical imaging, to form [ 18 F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [ 18 F]FDG was reacted with ß-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the α-1,4 and α-1,3-linked products 2-deoxy-[ 18 F]-fluoro-maltose ([ 18 F]FDM) and 2-deoxy-2-[ 18 F]-fluoro-sakebiose ([ 18 F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[ 18 F]fluoro-trehalose ([ 18 F]FDT), 2-deoxy-2-[ 18 F]fluoro-laminaribiose ([ 18 F]FDL), and 2-deoxy-2-[ 18 F]fluoro-cellobiose ([ 18 F]FDC). We subsequently tested [ 18 F]FDM and [ 18 F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. The lead sakebiose-derived tracer [ 18 F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [ 18 F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [ 18 F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.
ABSTRACT
From its structure and mechanism, sucrose phosphorylase is a specialized glycoside hydrolase that uses phosphate ions instead of water as the nucleophile of the reaction. Unlike the hydrolysis reaction, the phosphate reaction is readily reversible and, here, this has enabled the study of temperature effects on kinetic parameters to map the energetic profile of the complete catalytic process via a covalent glycosyl enzyme intermediate. Enzyme glycosylation from sucrose and α-glucose 1-phosphate (Glc1P) is rate-limiting in the forward (kcat = 84 s-1) and reverse direction (kcat = 22 s-1) of reaction at 30 °C. Enzyme-substrate association is driven by entropy (TΔSb ≥ +23 kJ/mol), likely arising from enzyme desolvation at the binding site for the leaving group. Approach from the ES complex to the transition state involves uptake of heat (ΔH⧧ = 72 ± 5.2 kJ/mol) with little further change in entropy. The free energy barrier for the enzyme-catalyzed glycoside bond cleavage in the substrate is much lower than that for the non-enzymatic reaction (knon), ΔΔG⧧ = ΔGnon⧧ - ΔGenzyme⧧ = +72 kJ/mol; sucrose. This ΔΔG⧧, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (â¼1014 M-1), is almost entirely enthalpic in origin. The enzymatic rate acceleration (kcat/knon) is â¼1012-fold and similar for reactions of sucrose and Glc1P. The 103-fold lower reactivity (kcat/Km) of glycerol than fructose in enzyme deglycosylation reflects major losses in the activation entropy, suggesting a role of nucleophile/leaving group recognition by the enzyme in inducing the active-site preorganization required for optimum transition state stabilization by enthalpic forces.
Subject(s)
Glucosyltransferases , Sucrose , Thermodynamics , Glucosyltransferases/metabolism , Phosphates/metabolism , Kinetics , CatalysisABSTRACT
As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine (U) by the corresponding C-nucleoside, pseudouridine (Ψ), in 1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me-Ψ-5'-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1N-5C rearrangement of ß-D-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via D-ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ-5'-phosphate (ΨMP), gives Ψ. Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the ΨMP, but not from the intermediary ribose phosphates. Discovery of Yjjg as ΨMP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N-C rearrangement, the optimized U reaction yields a supersaturated product solution (â¼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of â¼105 mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C-nucleoside synthesis in high efficiency.
Subject(s)
Nucleosides , Pseudouridine , Uridine/metabolism , Pseudouridine/metabolism , Biocatalysis , UracilABSTRACT
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.