3-hydroxyl Group Research Articles

Structure-guided discovery of bile acid derivatives for treating liver diseases without causing itch.

Chronic itch is a debilitating symptom profoundly impacting the quality of life in patients with liver diseases like cholestasis. Activation of the human G-protein coupled receptor, MRGPRX4 (hX4), by bile acids (BAs) is implicated in promoting cholestasis itch. However, the detailed underlying mechanisms remain elusive. Here, we identified 3-sulfated BAs that are elevated in cholestatic patients with itch symptoms. We solved the cryo-EM structure of hX4-Gq in a complex with 3-phosphated deoxycholic acid (DCA-3P), a mimic of the endogenous 3-sulfated deoxycholic acid (DCA-3S). This structure revealed an unprecedented ligand-binding pocket in MRGPR family proteins, highlighting the crucial role of the 3-hydroxyl (3-OH) group on BAs in activating hX4. Guided by this structural information, we designed and developed compound 7 (C7), a BA derivative lacking the 3-OH. Notably, C7 effectively alleviates hepatic injury and fibrosis in liver disease models while significantly mitigating the itch side effects.

Convergent synthesis of glycoalkaloids solasonine and its saponin derivative.

We present a practical and convergent synthesis of glycoalkaloids solasonine 1 and its saponin derivative 2, incorporating a {3-O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-β-D-galactopyranoside} moiety. The key features of this strategy include the following: (1) AuCl3-tBuCN cooperative catalysis enabling 1,2-trans stereoselective glycosidation of 2-branched trisaccharide trichloroacetimidate donors with steroidal aglycons, in the absence of neighboring group participation; (2) "cyanide effect" mediated regioselective benzoylation of the 4- and 6-hydroxyl groups of galactopyranosyl disaccharide; and (3) an effective approach to prevent orthoester byproduct formation.

Structural and biochemical insights into the molecular mechanism of N-acetylglucosamine/N-Acetylmuramic acid kinase MurK from Clostridium acetobutylicum

MurK is a MurNAc- and GlcNAc-specific amino sugar kinase, phosphorylates MurNAc and GlcNAc at the 6-hydroxyl group in an ATP-dependent manner, and contributes to the recovery of both amino sugars during the cell wall turnover in Clostridium acetobutylicum. Herein, we determined the crystal structures of MurK in complex with MurNAc, GlcNAc, and glucose, respectively. MurK represents the V-shaped fold, which is divided into a small N-terminal domain and a large C-terminal domain. The catalytic pocket is located within the deep cavity between the two domains of the MurK monomer. We mapped the significant enzyme-substrate interactions, identified key residues involved in the catalytic activity of MurK, and found that residues Asp77 and Arg78 from the β4-α2-loop confer structural flexibilities to specifically accommodate GlcNAc and MurNAc, respectively. Moreover, structural comparison revealed that MurK adopts closed-active conformation induced by the N-acetyl moiety from GlcNAc/MurNAc, rather than closed-inactive conformation induced by glucose, to carry out its catalytic reaction. Taken together, our study provides structural and functional insights into the molecular mechanism of MurK for the phosphorylation of both MurNAc and GlcNAc, sugar substrate specificity, and conformational changes upon sugar substrate binding.

Exploring the synthesis, structure and properties of two phenoxy-bridged polynuclear Cu(II) and Ni(II) complexes containing salamo-based bicompartmental ligand

A salamo-based bicompartmental ligand H4L was successfully prepared, which self-assembled with copper(II) and nickel(II) salts in stoichiometric ratios of 1:2 to construct polynuclear copper(II) and nickel(II) complexes: [Cu2(HL)(NO3)(H2O)] (1) and [Ni4(μ-HL)2(μ-OAc)2]⋅2CH3COCH3 (2). Complex 1 contains one partially deprotonated (HL)3− part, two copper(II) atoms, one coordinated monodentate nitrate ion and one coordinated water molecule. Each copper(II) center is located in the geometric structure of square pyramidal. While complex 2 is composed of four nickel(II) atoms, two ligand (HL)3− parts, and two bridging μ-OAc− anions. The differently structural complexes acquired may be attributed to the different solvents and anions used in the synthesis process. Whether or not 3-hydroxyl group of salicylaldehyde unit in H4L is involved in the coordination further influences the diversification of the structures of the complexes. The reaction sites and intermolecular interactions were further evaluated by MEPs, IRI, Hirshfeld surface and finally the fluorescence properties were explored.

Candidaantarctica Lipase B mediated kinetic resolution: A sustainable method for chiral synthesis of antiproliferative β-lactams

Biocatalysis is a valuable industrial approach in active pharmaceutical ingredient (API) manufacturing for asymmetric induction and synthesis of chiral APIs. Herein, we investigated synthesis of a panel of microtubule-destabilising antiproliferative β-lactam enantiomers employing a commercially available immobilised Candida antarctica lipase B enzyme together with methanol and MTBE. The β-lactam ring remained intact during chiral kinetic resolution reactions, plausibly due to a bulky N-1 phenyl substituent on the β-lactam ring substrate. The predominant reaction mediated by CAL-B was methanol catalysed conversion of the β-lactam 3-acetoxy substituent to a 3-hydroxyl group, with preferential methanolysis of the 3S, 4S enantiomer. The unreacted substrate underwent progressive enantioenrichment to the 3R, 4R enantiomer. Substitution patterns on the B ring C3 meta position of the β-lactam scaffold greatly affected the rate of reaction. Halo substituents (fluoro-, chloro- and bromo-) reduced the rate of conversion compared to unsubstituted analogues, which in turn increased enantiomeric excess (ee). Ee values up to 86 % for the 3S, 4S 3-hydroxyl enantiomer were achieved. A double resolution approach for unreacted substrate yielded high ee values (>99 %) for the 3R, 4R 3-acetoxy enantiomer. CAL-B mediated methanolysis is a more sustainable method for resolution of racemic antiproliferative β-lactams compared to a previous technique of chiral diastereomeric resolution. Yields of β-lactams obtained using CAL-B are far superior than previously described, which will facilitate progression toward pre-clinical and clinical development. Biocatalysis is a useful tool in the toolbox of the medicinal chemist.

Dual-cured organic–inorganic hard coating with high flexibility and transparency based on polysilsesquioxane and a functional polymer

Transparent hard-coating materials with glass-like wear resistance and plastic-like flexibility have attracted attention owing to their applicability in flexible/foldable displays. Materials with wear-resistant and bendable properties are rare because inorganic materials are normally hard and brittle, whereas organic materials, although flexible, typically lack wear resistance. This study entailed the development of a flexible hard coating, prepared by crosslinking a cycloaliphatic epoxy-functionalized oligosiloxane (CEOS) and hydroxylic-functionalized acrylic polymer with phthalic anhydride (PA) to form a highly crosslinked organic–inorganic coating network. CEOS, a UV-curable high-hardness inorganic base coating material, was synthesized by a sol–gel reaction. Thermally curable acrylic polymer and PA as a crosslinker were then introduced to simultaneously improve flexibility and hardness. Two differently curable materials were blended to maximize the coating properties through the synergistic effect of the different curing characteristics. To optimize the curing system, a dual-curing process involving UV irradiation and heat treatment was introduced. In the first step, UV irradiation initiated the ring-opening polymerization of the epoxide rings of CEOS. In the second step, the epoxide ring of CEOS, the hydroxylic group of the acrylic polymer resin, and the anhydride group of PA were thermally cured, and a multi-crosslinked network coating film was fabricated. The prepared coating exhibited high hardness (nano-indentation hardness of 0.63 GPa and a pencil hardness of up to 8H) and outstanding optical transparency (up to 91 % at 500 nm). The coated layer on the PET film exhibited high flexibility and could be bent to form a U-shaped cross-section without coating damage.

Polystyrene degradation by bacteria isolated from the larvae of <i>Rhynchophorusphoenicis</i>

The larvae of insects of the order Coleoptera have been reported to biodegrade plastics aided by their chewing mouthparts and the activities of their gut biota. However, there is no report of this ability by the African palm weevil (Rhynchophorus phoenicis). This study aims to the ability of R. phoenicis larvae to biodegrade polystyrene (PS). A total of 100 R. phoenicis larvae were fed for 21 days with PS foam, and afterwards, the gut contents of survivors were investigated for possible PS-degrading bacteria. Bacterial isolates were screened for PS biodegradation in an Erlenmeyer flask with PS film as the sole carbon source, in a mineral salt medium (MSM) at a temperature of 30oC and a pH of 7, for a period of 28 days. The isolates were used for biodegradation assay under the same conditions, for 60 days. The weight of PS films was determined before and after the biodegradation assay. Chemical changes in the films were confirmed by Fourier Transform Infrared (FTIR) spectroscopy. Two bacterial isolates were recovered from the gut of the only surviving R. phoenicis larvae fed with 100% PS. The isolates were identified based on their 16S rRNA sequences as Lysinibacillus macriodes and Pantoea dispersa with accession numbers OQ652017 and OQ652023 respectively. The isolates caused an 8.8% reduction in the weight of PS film and FTIR spectroscopy results confirmed the formation of groups suggestive of degradation products with the carbonyl group showing up as absorption peaks in the range of 1640-1760 cm-1 and the hydroxylic group at 3000-3700 cm-1 . The isolates were able to produce polyhydroxyalkanoate (PHA) equivalent to 1.4g/L, under PS degradation conditions. Therefore, coupling the biodegradation of PS with PHAproduction could be useful for the valorization of PS waste.

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity.

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Chemical and chemoenzymatic syntheses of sialyl Lewisa tetrasaccharide antigen.

Sialyl Lewisa (sLea), also known as cancer antigen 19-9, is a tumor-associated carbohydrate antigen. In this article, chemical and chemoenzymatic syntheses of a tetrasaccharide glycan 1 structurally derived from sLea are reported. Challenges involved in the chemical synthesis include the highly stereoselective construction of 1,2-cis-α-L-fucoside and α-D-sialoside, as well as the assembly of the 3,4-disubstituted N-acetylglucosamine subunit. Perbenzylated thiofucoside and N-acetyl-5-N,4-O-oxazolidinone protected sialic acid thioglycoside were employed as glycosyl donors, respectively, for the efficient preparation of the desired α-fucoside and α-sialoside. The 3,4-branched glucosamine backbone was established through a 3-O and then 4-O glycosylation sequence in which the 3-hydroxyl group of the glucosamine moiety was glycosylated first and then the 4-hydroxyl. A facile chemoenzymatic approach was also exploited to synthesize the target molecule. The chemically obtained free disaccharide 30 was sequentially sialylated and fucosylated in an enzyme-catalyzed regio- and stereospecific manner to form 1 in high yields. The linker appended 1 can be covalently attached to a carrier protein for further immunological studies.

Molecular mechanism of calcitriol enhances membrane water permeability

Helicobacter pylori (H. pylori) exhibits a unique membrane lipid composition, including dimyristoyl phosphatidylethanolamine (DMPE) and cholesterol, unlike other Gram-negative bacteria. Calcitriol has antimicrobial activity against H. pylori, but cholesterol enhances antibiotics resistance in H. pylori. This study explored the changes in membrane structure and the molecular mechanisms of cholesterol/calcitriol translocation using well-tempered metadynamics (WT-MetaD) simulations and microsecond conventional molecular dynamics (CMD) simulations. Calcitriol facilitated water transport across the membrane, while cholesterol had the opposite effect. The differing effects might result from the tail 25-hydroxyl group and a wider range of orientations of calcitriol in the DMPE/dimyristoyl phosphatidylglycerol (DMPG) (3:1) membrane. Calcitriol moves across the bilayer center without changing its orientation along the membrane Z-axis, becomes parallel to the membrane surface at the membrane-water interface, and then rotates approximately 90° in this interface. The translocation mechanism of calcitriol is quite different from the flip-flop of cholesterol. Moreover, calcitriol crossed from one layer to another more easily than cholesterol, causing successive perturbations to the hydrophobic core and increasing water permeation. These results improve our understanding of the relationship between cholesterol/calcitriol concentrations and the lipid bilayer structure and the role of lipid composition in water permeation.

A UPLC-based activity assay method for aminoglycoside phosphotransferase 4-Ia (APH4): A Selectable marker of plant transformation

The aminoglycoside phosphotransferase 4-Ia (APH4) is a hygromycin B phosphotransferase and catalyzes the phosphorylation of the 4-hydroxyl group of the antibiotic hygromycin B, inactivating its antibiotic activity. Therefore, APH4 has utility as a selectable marker for transformation of many plant species. However, suitable methods to measure and quantify plants expressing APH4 enzymes are lacking because of technical challenges, associated with both specificity and sensitivity. Here we describe a highly specific and sensitive APH4 enzymatic activity assay by measuring the production of adenosine 5′-diphosphate (ADP) using ultra performance liquid chromatography (UPLC)-based method. The present assay enabled determination of enzymatic activity of APH4 over a concentration range from 0.05 to 0.8 μg APH4/ml. Method performance validation sample were examined at five concentrations in triplicate in six independent sessions. Average CVs of 5.1 % for intra-assay and 11.7 % for inter-assay over all samples were determined for precision, and 6.0 % was determined for accuracy. Additionally, the method was validated for use to determine APH4 enzymatic activity in both microbially produced and plant-derived samples.

Separation of Enantiomers of Metolazone by Thin Layer Chromatography Using a Chiral Selector

Enantiomeric separation of (RS)-Metolazone has been achieved by thin-layer chromatography (TLC) using both direct modes.  cyclodextrin, containing hydroxylic group, was utilized as the chiral selector. For enantioseparation, cyclodextrin was used as a chiral additive in the stationary phase in a non-covalent mode and there was no chiral additive in the mobile phase; the native enantiomers were isolated and characterized. Cyclodextrin was also added to mobile phase and there was no chiral selector in the stationary phase. The spots were then isolated and characterized. The effect of the composition of the mobile phase, pH, and temperature was studied for the optimization of successful separation conditions. The spots were located in an iodine chamber.

Design, synthesis, and anti-respiratory syncytial virus potential of novel 3-(1,2,3-triazol-1-yl)furoxazine-fused benzimidazole derivatives

Respiratory syncytial virus (RSV) is a major cause of serious lower respiratory tract infections in infants, children, and older persons. Currently, the only approved anti-viral chemotherapeutic drug for RSV treatment is ribavirin aerosol; however, its significant toxicity has led to restricted clinical use. In a previous study, we developed various benzimidazole derivatives against RSV. In this study, we synthesised 3-azide substituted furoxazine-fused benzimidazole derivatives by sulfonylation and azide substitution of the 3-hydroxyl group of the furoxazine-fused benzimidazole derivatives. Subsequently, a series of 3-(1,2,3-triazol-1-yl)-substituted furoxazine-fused benzimidazole derivatives were synthesised using the classical click reaction. Biological evaluations of the target compounds indicated that compound 4a-2 had higher activity against RSV (EC50 = 12.17 μM) and lower cytotoxicity (CC50 = 390.64 μM). Compound 4a-2 exerted anti-viral effects against the RSV Long strain by inhibiting apoptosis and the elevation of reactive oxygen species (ROS) and inflammatory factors caused by viral infection in vitro. Additionally, the clinical symptoms of the virus-infected mice were markedly relieved, and the viral load in the lung tissues was dramatically decreased. The biosafety profile of compound 4a-2 was also favourable, showing no detectable adverse effects on any of the major organs in vivo. These findings underscore the potential of compound 4a-2 as a valuable therapeutic option for combating RSV infections while also laying the foundation for further research and development in the field.

Electrochemical oxidation and superoxide radical scavenging activity of 2-hydroxy/methoxy-phenylbenzothiazole derivatives

Electrochemical characterisation and antioxidant activity against superoxide radical anion of four 2-hydroxy/methoxy-phenylbenzothiazole derivatives (1–4) are reported.The electrochemical oxidation was studied at a glassy carbon electrode using square-wave voltammetry in an aqueous buffer solution (pH = 1–12). In 1 (2-(2-methoxyphenyl)benzothiazole-6-ammonium chloride) and 2 (2-(2-hidroxyphenyl)benzothiazole-6-ammonium chloride), the benzothiazole C6-amino group was the main electroactive centre, undergoing a complex, pH-dependent process, coupled to the follow-up chemical reaction. The initial electrode reaction of 3 (6-(4,5-dihydro-1H-imidazol-3-ium-2-yl)-2-(2,3,4-trihydroxyphenyl)benzothiazole chloride) and 4 (6-amidinium-2-(2,3,4-trihydroxyphenyl)benzothiazole chloride) is a quasireversible, two-electrons, two-protons oxidation of the pyrogallol group.Superoxide radical scavenging activity was evaluated by means of cyclic voltammetry and computational analysis, which was revealed to rely on their ability to undergo the hydrogen atom transfer. In 1 and 2, H• donation from the benzothiazole C6-amino group is favoured, while a considerable improvement in the antioxidant activity was achieved in systems with the pyrogallol moiety 3 and 4, where it is realized from the middle 2-hydroxyl group, which allows for the formation of a stable OH∙∙∙O•∙∙∙HO fragment following the H• liberation.

Design of a chimeric glycosyltransferase OleD for the site-specific O-monoglycosylation of 3-hydroxypyridine in nosiheptide.

To identify the potential role of the 3-hydroxyl group of the pyridine ring in nosiheptide (NOS) for its antibacterial activity against Gram-positive pathogens, enzymatic glycosylation was utilized to regio-selectively create a monoglycosyl NOS derivative, NOS-G. For this purpose, we selected OleD, a UDP glycosyltransferase from Streptomyces antibioticus that has a low productivity for NOS-G. Activity of the enzyme was increased by swapping domains derived from OleI, both single and in combination. Activity enhancement was best in mutant OleD-10 that contained four OleI domains. This chimer was engineered by site-directed mutagenesis (single and in combination) to increase its activity further, whereby variants were screened using a newly-established colorimetric assay. OleD-10 with I117F and T118G substitutions (FG) had an increased NOS-G productivity of 56%, approximately 70 times higher than that of wild-type OleD. The reason for improved activity of FG towards NOS was structurally attributed to a closer distance (<3 Å) between NOS/sugar donor and the catalytic amino acid H25. The engineered enzyme allowed sufficient activity to demonstrate that the produced NOS-G had enhanced stability and aqueous solubility compared to NOS. Using a murine MRSA infection model, it was established that NOS-G resulted in partial protection within 20 h of administration and delayed the death of infected mice. We conclude that 3-hydroxypyridine is a promising site for structural modification of NOS, which may pave the way for producing nosiheptide derivatives as a potential antibiotic for application in clinical treatment.

Structural and Biochemical Analysis of 3-Dehydroquinate Dehydratase from Corynebacterium glutamicum.

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQDWT with the citrate at a resolution of 1.80Å and CgDHQDR19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQDS103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQDS103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

Enaminoketones - functional derivatives based on 7-hydroxy-3′,3′-dimethyl-3′&lt;i&gt;H&lt;/i&gt;-spiro[chromen-2,1′-isobenzofuran]-8-carbaldehyde with aromatic amines. physicochemical studies and biological activity

This research focuses on studying derivatives of 2-oxaindane spiropyran by its condensation with aromatic amines. The results showed that all the synthesized compounds are enaminoketones form in both solution and solid state. This is explained by the fact that during the formation of Schiff bases, the proton of the 7-hydroxyl group of the benzopyran part migrates to the azomethine nitrogen, which leads to the transition of the cyclic form of spiropyran into the corresponding enaminoketone. In the DMSO solution, the enaminoketones dynamic equilibrium of the E , Z -isomeric forms were observed. The structure of enaminoketone based on 3,4-dimethylaniline was proved by the X-ray diffraction method. The synthesized compounds were studied for their in vitro anticancer activity against human hepatocellular carcinoma (HepG2), breast carcinoma (MCF-7), lung cancer (A549), and carcinoma cells (KB). Enaminoketone with R = 3,4-СH3Ph is characterized by the highest activity, with IC50 values of 13.38 μM (KB). The results in vitro antioxidant activity test using 1,1-diphenyl-2-picrylhydrazyl (DPPH) indicated that enaminoketones were inactive.

Identification of new 4-(6-oxopyridazin-1-yl)benzenesulfonamides as multi-target anti-inflammatory agents targeting carbonic anhydrase, COX-2 and 5-LOX enzymes: synthesis, biological evaluations and modelling insights

Multiple inhibitions of CA, COX-2 and 5-LOX enzymes has been recognised as a useful strategy for the development of anti-inflammatory drugs that can avoid the disadvantages of using NSAIDs alone. Here, we report new pyridazine-based sulphonamides (5a-c and 7a-f) as potential multi-target anti-inflammatory candidates. First, the furanone heterocycle in the dual CA/COX-2 inhibitor Polmacoxib was replaced with the pyridazinone one. Then, a hydrophobic tail was appended through benzylation of the 3-hydroxyl group of the pyridazinone scaffold to afford benzyloxy pyridazines 5a-c. Furthermore, the structures were adorned with the polar sulphonate functionality, in pyridazine sulphonates 7a-f, that are expected to be engaged in interactions with the hydrophilic half of the CA binding sites. All of the disclosed pyridazinones were tested for inhibitory activities against 4 hCA isoforms (I, II, IX, and XII), as well as against COX-1/2, and 5-LOX. Furthermore, in vivo anti-inflammatory and analgesic effects of pyridazinones 7a and 7b were examined.

Structural analysis and biological activities of C25-amino and C25-nitro vitamin D analogs

Intense synthetic efforts have been directed towards the development of noncalcemic analogs of 1,25-dihydroxyvitamin D3. We describe here the structural analysis and biological evaluation of two derivatives of 1,25-dihydroxyvitamin D3 with modifications limited to the replacement of the 25-hydroxyl group by a 25-amino or 25-nitro groups. Both compounds are agonists of the vitamin D receptor. They mediate biological effects similar to 1,25-dihydroxyvitamin D3, the 25-amino derivative being the most potent one while being less calcemic than 1,25-dihydroxyvitamin D3. The in vivo properties of the compounds make them of potential therapeutic value.

Substrate-Assisted Mechanism for the Degradation of N-Glycans by a Gut Bacterial Mannoside Phosphorylase

The unknown human gut bacterium mannoside phosphorylase (UhgbMP) is involved in the metabolization of eukaryotic N-glycans lining the intestinal epithelium, a factor associated with the onset and symptoms of inflammatory bowel diseases. In contrast with most glycoside phosphorylases, the putative catalytic acid of UhgbMP, Asp104, is far from the scissile glycosidic bond, challenging the classical Koshland mechanism. Using quantum mechanics/molecular mechanics metadynamics, we demonstrate that the enzyme operates by substrate-assisted catalysis via the 3-hydroxyl group of the mannosyl unit, following a 1S5/B2,5 → [B2,5]‡ → 0S2 conformational itinerary. Given the conservation of the active site hydrogen bond network across the family, this mechanism is expected to apply to other GH130 enzymes, as well as recently characterized mannoside phosphorylases with similar folds. Gaining insight into the catalytic reaction of these enzymes can aid the design of specific inhibitors to control interactions between gut microbes and the host.