Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Chemical Synthesis of a Densely Functionalized Aminodisaccharide from Fusobacterium nucleatum ATCC 23726 O-Antigen.

Fusobacterium nucleatum, a colorectal-cancer-associated oncomicrobe, can trigger or accelerate numerous pathologies. We report the first synthesis of a conjugation-ready disaccharide containing six amino groups from F. nucleatum ATCC 23726 O-antigen. Rare 2,3-diamido-d-glucuronic acid amide and 2-acetamido-4-amino-d-fucose were synthesized from d-glucosamine through configuration inversion, nucleophilic substitution, C6 oxidation, and C6 deoxygenation. A judicious choice of protecting groups and reaction conditions enabled the selective installation of N-acetyl, N-propanoyl, N-formyl, and carboxamido groups.

Two populations of protein molecules detected by small-angle neutron and X-ray scattering (SANS and SAXS) in lyophilized protein:lyoprotector (disaccharide) systems.

Two protein interaction peaks are observed in pharmaceutically-relevant protein (serum albumin) : disaccharide 1 : 1 and 1 : 3 (w/w) freeze-dried systems for the first time. In samples with a higher disaccharide content, the protein-protein distances are longer for both populations, while the fraction of the protein population with a shorter protein-protein distance is lower. Both factors would favor better stability against aggregation for disaccharide-rich protein formulations. This study provides direct experimental support for a "dilution" hypothesis as a potential stabilization mechanism for freeze-dried protein formulations.

Rutinosides-derived from Sarocladium strictum 6-O-α-rhamnosyl-ß-glucosidase show enhanced anti-tumoral activity in pancreatic cancer cells.

BACKGROUND: Low targeting efficacy and high toxicity continue to be challenges in Oncology. A promising strategy is the glycosylation of chemotherapeutic agents to improve their pharmacodynamics and anti-tumoral activity. Herein, we provide evidence of a novel approach using diglycosidases from fungi of the Hypocreales order to obtain novel rutinose-conjugates therapeutic agents with enhanced anti-tumoral capacity. RESULTS: Screening for diglycosidase activity in twenty-eight strains of the genetically related genera Acremonium and Sarocladium identified 6-O-α-rhamnosyl-ß-glucosidase (αRßG) of Sarocladium strictum DMic 093557 as candidate enzyme for our studies. Biochemically characterization shows that αRßG has the ability to transglycosylate bulky OH-acceptors, including bioactive compounds. Interestingly, rutinoside-derivatives of phloroglucinol (PR) resorcinol (RR) and 4-methylumbelliferone (4MUR) displayed higher growth inhibitory activity on pancreatic cancer cells than the respective aglycones without significant affecting normal pancreatic epithelial cells. PR exhibited the highest efficacy with an IC50 of 0.89 mM, followed by RR with an IC50 of 1.67 mM, and 4MUR with an IC50 of 2.4 mM, whereas the respective aglycones displayed higher IC50 values: 4.69 mM for phloroglucinol, 5.90 mM for resorcinol, and 4.8 mM for 4-methylumbelliferone. Further, glycoconjugates significantly sensitized pancreatic cancer cells to the standard of care chemotherapy agent gemcitabine. CONCLUSIONS: αRßG from S. strictum transglycosylate-based approach to synthesize rutinosides represents a suitable option to enhance the anti-proliferative effect of bioactive compounds. This finding opens up new possibilities for developing more effective therapies for pancreatic cancer and other solid malignancies.

Receptor Targeting Using Copolymer-Modified Gold Nanoparticles for pCMV-Luc Gene Delivery to Liver Cancer Cells In Vitro.

The formulation of novel delivery protocols for the targeted delivery of genes into hepatocytes by receptor mediation is important for the treatment of liver-specific disorders, including cancer. Non-viral delivery methods have been extensively studied for gene therapy. Gold nanoparticles (AuNPs) have gained attention in nanomedicine due to their biocompatibility. In this study, AuNPs were synthesized and coated with polymers: chitosan (CS), and polyethylene glycol (PEG). The targeting moiety, lactobionic acid (LA), was added for hepatocyte-specific delivery. Physicochemical characterization revealed that all nano-formulations were spherical and monodispersed, with hydrodynamic sizes between 70 and 250 nm. Nanocomplexes with pCMV-Luc DNA (pDNA) confirmed that the NPs could bind, compact, and protect the pDNA from nuclease degradation. Cytotoxicity studies revealed that the AuNPs were well tolerated (cell viabilities > 70%) in human hepatocellular carcinoma (HepG2), embryonic kidney (HEK293), and colorectal adenocarcinoma (Caco-2) cells, with enhanced transgene activity in all cells. The inclusion of LA in the NP formulation was notable in the HepG2 cells, which overexpress the asialoglycoprotein receptor on their cell surface. A five-fold increase in luciferase gene expression was evident for the LA-targeted AuNPs compared to the non-targeted AuNPs. These AuNPs have shown potential as safe and suitable targeted delivery vehicles for liver-directed gene therapy.

Synthesis of Sucrose-Mimicking Disaccharide by Intramolecular Aglycone Delivery.

Rare sugars are known for their ability to suppress postprandial blood glucose levels. Therefore, oligosaccharides and disaccharides derived from rare sugars could potentially serve as functional sweeteners. A disaccharide [α-d-allopyranosyl-(1→2)-ß-d-psicofuranoside] mimicking sucrose was synthesized from rare monosaccharides D-allose and D-psicose. Glycosylation using the intermolecular aglycon delivery (IAD) method was employed to selectively form 1,2-cis α-glycosidic linkages of the allopyranose residues. Moreover, ß-selective psicofuranosylation was performed using a psicofuranosyl acceptor with 1,3,4,6-tetra-O-benzoyl groups. This is the first report on the synthesis of non-reducing disaccharides comprising only rare d-sugars by IAD using protected ketose as a unique acceptor; additionally, this approach is expected to be applicable to the synthesis of functional sweeteners.

Exploring long-range fluorine-carbon J-coupling for conformational analysis of deoxyfluorinated disaccharides: A combined computational and NMR study.

In this study, we investigated the potential of long-range fluorine-carbon J-coupling for determining the structures of deoxyfluorinated disaccharides. Three disaccharides, previously synthesized as potential galectin inhibitors, exhibited through-space fluorine-carbon J-couplings. In our independent conformational analysis of these disaccharide derivatives, we employed a combination of density functional theory (DFT) calculations and nuclear magnetic resonance (NMR) experiments. By comparing the calculated nuclear shieldings with the experimental carbon chemical shifts, we were able to identify the most probable conformers for each compound. A model comprising fluoromethane and methane molecules was used to study the relationship between molecular arrangements and intermolecular through-space J-coupling. Our study demonstrates the important effect of internuclear distance and molecular orientation on the magnitude of fluorine-carbon coupling. The experimental values for the fluorine-carbon through-space couplings (TSCs) of the disaccharides corresponded with values calculated for the most probable conformers identified by the conformational analysis. These results unlock the broader application of fluorine-carbon TSCs as powerful tools for conformational analysis of flexible molecules, offering valuable insights for future structural investigations.

Structural Characterization of Disaccharides Using Cyclic Ion Mobility Spectrometry and Monosaccharide Standards.

To understand the mode of action of bioactive oligosaccharides, such as prebiotics, in-depth knowledge about all structural features, including monosaccharide composition, linkage type, and anomeric configuration, is necessary. Current analytical techniques provide limited information about structural features within complex mixtures unless preceded by extensive purification. In this study, we propose an approach employing cyclic ion mobility spectrometry (cIMS) for the in-depth characterization of oligosaccharides, here demonstrated for disaccharides. We were able to separate galactose and glucose anomers by exploiting the high ion mobility resolution of cIMS. Using the obtained monosaccharide mobilograms as references, we determined the composition and anomeric configuration of 4ß-galactobiose by studying the monosaccharide fragments generated by collision-induced dissociation (CID) before the ion mobility separation. Drift times and individual MS2 spectra of partially resolved reducing-end anomers of 4ß-galactobiose, 4ß-galactosylglucose (lactose), and 4ß-glucosylglucose (cellobiose) were obtained by deconvolution using CID fragmentation induced in the transfer region between the cIMS cell and TOF analyzer. The composition and anomeric configuration of the reducing end anomers of these disaccharides were identified using cIMS2 approaches, where first each anomer was isolated using cIMS and individually fragmented, and the monosaccharide fragments were again separated by cIMS for comparison with monosaccharide standards. With these results we demonstrate the promising application of cIMS for the structural characterization of isomeric oligosaccharides.

Activation of glycosyl methylpropiolates by TfOH.

Activation of glycosyl methylpropiolates by TfOH was investigated. Armed and superarmed glycosyl donors can be activated by use of 0.2 equivalent TfOH whereas 1.0 equivalent of TfOH was required for the activation of the disarmed glycosyl donors. All the glycosidations gave very good yields. The method is suitable for synthesis of glycosides and disaccharides and it may result in the hydrolysis of the interglycosidic bond if the sugar at the non-reducing end is armed or superarmed. These problems are not seen when gold-catalyzed activation procedures are invoked for the activation of glycosyl alkynoates.

Does Inter-Residue Hydrogen Bonding in ß-(1→4)-Linked Disaccharides Influence Linkage Conformation in Aqueous Solution?

Two disaccharides, methyl ß-d-galactopyranosyl-(1→4)-α-d-glucopyranoside (1) and methyl ß-d-galactopyranosyl-(1→4)-3-deoxy-α-d-ribo-hexopyranoside (3), were prepared with selective 13C-enrichment to allow measurement of six trans-O-glycosidic J-couplings (2JCOC, 3JCOCH, and 3JCOCC) in each compound. Density functional theory (DFT) was used to parameterize Karplus-like equations that relate these J-couplings to either ϕ or ψ. MA'AT analysis was applied to both linkages to determine mean values of ϕ and ψ in each disaccharide and their associated circular standard deviations (CSDs). Results show that deoxygenation at C3 of 1 has little effect on both the mean values and librational motions of the linkage torsion angles. This finding implies that, if inter-residue hydrogen bonding between O3H and O5' of 1 is present in aqueous solution and persistent, it plays little if any role in dictating preferred linkage conformation. Hydrogen bonding may lower the energy of the preferred linkage geometry but does not determine it to any appreciable extent. Aqueous 1-µs MD simulation supports this conclusion and also indicates greater conformational flexibility in deoxydisaccharide 3 in terms of sampling several, conformationally distinct, higher-energy conformers in solution. The populations of these latter conformers are low (3-14%) and could not be validated by MA'AT analysis. If the MD model is correct, however, C3 deoxygenation does enable conformational sampling over a wider range of ϕ/ψ values, but linkage conformation in the predominant conformer is essentially identical in both 1 and 3.

Degradation Mechanisms of Six Typical Glucosidic Bonds of Disaccharides Induced by Free Radicals.

Increasing hydrogen peroxide (H2O2)-based systems have been developed to degrade various polysaccharides due to the presence of highly reactive free radicals, but published degradation mechanisms are still limited. Therefore, this study aimed to clarify the degradation mechanism of six typical glucosidic bonds from different disaccharides in an ultraviolet (UV)/H2O2 system. The results showed that the H2O2 concentration, disaccharide concentration, and radiation intensity were important factors affecting pseudo-first-order kinetic constants. Hydroxyl radical, superoxide radical, and UV alone contributed 58.37, 18.52, and 19.17% to degradation, respectively. The apparent degradation rates ranked in the order of cellobiose ≈ lactose > trehalose ≈ isomaltose > turanose > sucrose ≈ maltose. The reaction pathways were then deduced after identifying their degradation products. According to quantum chemical calculations, the cleavage of α-glycosidic bonds was more kinetically unfavorable than that of ß-glycosidic bonds. Additionally, the order of apparent degradation rates depended on the energy barriers for the formation of disaccharide-based alkoxyl radicals. Moreover, energy barriers for homolytic scissions of glucosidic C1-O or C7-O sites of these alkoxyl radicals ranked in the sequence: α-(1 → 2) ≈ α-(1 → 3) < α-(1 → 4) < ß-(1 → 4) < α-(1 → 6) < α-(1 → 1) glucosidic bonds. This study helps to explain the mechanisms of carbohydrate degradation by free radicals.

Conformational Preferences at the Glycosidic Linkage of Saccharides in Solution as Deduced from NMR Experiments and MD Simulations: Comparison to Crystal Structures.

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles φ and ψ have been investigated for O-methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in α-l-Fucp-(1→3)-ß-d-Glcp-OMe, α-l-Fucp-(1→3)-α-d-Galp-OMe and α-d-Glcp-(1→4)-α-d-Galp-OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one-dimensional 1 H,1 H-NOESY NMR experiments to obtain transglycosidic proton-proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3 JCH transglycosidic coupling constants related to torsion angles φ and ψ. Computed effective proton-proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the α-(1→3)-linked disaccharides and revealed that for the bimodal distribution at the ψ torsion angle for the α-(1→4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter-residue hydrogen bonding pattern for the α-(1→4)-linked disaccharide, including a nonconventional hydrogen bond between H5' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1 H NMR chemical shift displacement compared to α-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles φ and ψ in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation.

Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Biological properties of (1-4)-thio disaccharides.

Thio sugars are carbohydrate derivatives in which one or more oxygen atoms have been replaced with sulfur. Thio sugars are effective inhibitors of glycosylases, have considerable therapeutic potential, and are used as drugs in the treatment of diabetes and infectious diseases. The development of this branch of carbohydrate chemistry would not be possible without the development of novel methods for its synthesis and the analysis of their biochemical properties. In this Review Article, we summarize our findings on the biological properties of a collection of thio sugars and their derivatives synthesized by the Witczak and Bielski team using their original methods based on the Michael addition of sugar thiols to levoglucosenone.

The collision-induced dissociation mechanism of sodiated Hex-HexNAc disaccharides.

Determining carbohydrate structures, such as their compositions, linkage positions, and in particular the anomers and stereoisomers, is a great challenge. Isomers of different anomers or stereoisomers have the same sequences of chemical bonds, but have different orientations of some chemical bonds which are difficult to be distinguished by mass spectrometry. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is a widely used technique for characterizing carbohydrate structures. Understanding the carbohydrate dissociation mechanism is important for obtaining the structural information from MS/MS. In this work, we studied the CID mechanism of galactose-N-acetylgalactosamine (Gal-GalNAc) and glucose-N-acetylglucosamine (Glc-GlcNAc) disaccharides with 1→3 and 1→4 linkages. For Gal-GalNAc disaccharides, the CID mass spectra of sodium ion adducts show significant difference between the α- and ß-anomers of GalNAc at the reducing end, while no difference in the CID mass spectra between two anomers of Glc-GlcNAc disaccharides was found. Quantum chemistry calculations show that for Gal-GalNAc disaccharides, the difference of the dissociation barriers between dehydration and glycosidic bond cleavage is significantly small in the ß-anomer compared to that in the α-anomer; while these differences are similar between the α- and ß-anomers of Glc-GlcNAc disaccharides. These differences can be attributed to the different orientations of hydroxyl and N-acetyl groups located at GalNAc and GlcNAc. The calculation results are consistent with the CID spectra of isotope labelled disaccharides. Our study provides an insight into the CID of 1→3 and 1→4 linked Gal-GalNAc and Glc-GlcNAc disaccharides. This information is useful for determining the anomeric configurations of GalNAc in oligosaccharides.

New insights into the protein stabilizing effects of trehalose by comparing with sucrose.

Disaccharides are well known to be efficient stabilizers of proteins, for example in the case of lyophilization or cryopreservation. However, although all disaccharides seem to exhibit bioprotective and stabilizing properties, it is clear that trehalose is generally superior compared to other disaccharides. The aim of this study was to understand this by comparing how the structural and dynamical properties of aqueous trehalose and sucrose solutions influence the protein myoglobin (Mb). The structural studies were based on neutron and X-ray diffraction in combination with empirical potential structure refinement (EPSR) modeling, whereas the dynamical studies were based on quasielastic neutron scattering (QENS) and molecular dynamics (MD) simulations. The results show that the overall differences in the structure and dynamics of the two systems are small, but nevertheless there are some important differences which may explain the superior stabilizing effects of trehalose. It was found that in both systems the protein is preferentially hydrated by water, but that this effect is more pronounced for trehalose, i.e. trehalose forms less hydrogen bonds to the protein surface than sucrose. Furthermore, the rotational motion around dihedrals between the two glucose rings of trehalose is slower than in the case of the dihedrals between the glucose and fructose rings of sucrose. This leads to a less perturbed protein structure in the case of trehalose. The observations indicate that an aqueous environment closest to the protein molecules is beneficial for an efficient bioprotective solution.

Probing Disaccharide Binding to Triplatin as Models for Tumor Cell Heparan Sulfate (GAG) Interactions.

In this study, we have used [1H, 15N] NMR spectroscopy to investigate the interactions of the trinuclear platinum anticancer drug triplatin (1) (1,0,1/t,t,t or BBR3464) with site-specific sulfated and carboxylated disaccharides. Specifically, the disaccharides GlcNS(6S)-GlcA (I) and GlcNS(6S)-IdoA(2S) (II) are useful models of longer-chain glycosaminoglycans (GAGs) such as heparan sulfate (HS). For both the reactions of 15N-1 with I and II, equilibrium conditions were achieved more slowly (65 h) compared to the reaction with the monosaccharide GlcNS(6S) (9 h). The data suggest both carboxylate and sulfate binding of disaccharide I to the Pt with the sulfato species accounting for <1% of the total species at equilibrium. The rate constant for sulfate displacement of the aqua ligand (kL2) is 4 times higher than the analogous rate constant for carboxylate displacement (kL1). There are marked differences in the equilibrium concentrations of the chlorido, aqua, and carboxy-bound species for reactions with the two disaccharides, notably a significantly higher concentration of carboxylate-bound species for II, where sulfate-bound species were barely detectable. The trend mirrors that reported for the corresponding dinuclear platinum complex 1,1/t,t, where the rate constant for sulfate displacement of the aqua ligand was 3 times higher than that for acetate. Also similar to what we observed for the reactions of 1,1/t,t with the simple anions, aquation of the sulfato group is rapid, and the rate constant k-L2 is 3 orders of magnitude higher than that for displacement of the carboxylate (k-L1). Molecular dynamics calculations suggest that extra hydrogen-bonding interactions with the more sulfated disaccharide II may prevent or diminish sulfate binding of the triplatin moiety. The overall results suggest that Pt-O donor interactions should be considered in any full description of platinum complex cellular chemistry.

Molecular implications of glycosaminoglycans in diabetes pharmacotherapy.

Diabetes mellitus causes a wide range of metabolic derangements with multiple organ damage. The microvascular and macrovascular complications of diabetes result partly from the damage to the glycosaminoglycans (GAG) in the basement membrane. GAGs are negatively charged polysaccharides with repeating disaccharide units. They play a significant role in cellular proliferation and signal transduction. Destruction of extracellular matrix results in diseases in various organs including myocardial fibrosis, retinal damage and nephropathy. To substitute the natural GAGs pharmacotherapeutically, they have been synthesized by using basic disaccharide units. Among the four classes of GAGs, heparin is the most widely studied. Recent studies have revealed multiple significant GAG-protein interactions suggesting their use for the management of diabetic complications. Moreover, they can act as biomarkers for assessing the disease progression. A number of GAG-based therapeutic agents are being evaluated for managing diabetic complications. The current review provides an outline of the role of GAGs in diabetes while covering their interaction with different molecular players that can serve as targets for the diagnosis, management and prevention of diabetes and its complications. The medicinal chemistry and clinical pharmacotherapeutics aspects have are covered to aid in the establishment of GAG-based therapies as a possible avenue for diabetes.

Enzymatic comparison of two homologous enzymes reveals N-terminal domain of chondroitinase ABC I regulates substrate selection and product generation.

Chondroitinase ABC-type I (CSase ABC I), which can digest both chondroitin sulfate (CS) and dermatan sulfate (DS) in an endolytic manner, is an essential tool in structural and functional studies of CS/DS. Although a few CSase ABC I have been identified from bacteria, the substrate-degrading pattern and regulatory mechanisms of them have rarely been investigated. Herein, two CSase ABC I, IM3796 and IM1634, were identified from the intestinal metagenome of CS-fed mice. They show high sequence homology (query coverage: 88.00%, percent identity: 90.10%) except for an extra peptide (Met1-His109) at the N-terminus in IM1634, but their enzymatic properties are very different. IM3796 prefers to degrade 6-O-sulfated GalNAc residue-enriched CS into tetra- and disaccharides. In contrast, IM1634 exhibits nearly a thousand times more activity than IM3796 and can completely digest CS/DS with various sulfation patterns to produce disaccharides, unlike most CSase ABC I. Structure modeling showed that IM3796 did not contain an N-terminal domain composed of two ß-sheets, which is found in IM1634 and other CSase ABC I. Furthermore, deletion of the N-terminal domain (Met1-His109) from IM1634 caused the enzymatic properties of the variant IM1634-T109 to be similar to those of IM3796, and conversely, grafting this domain to IM3796 increased the similarity of the variant IM3796-A109 to IM1634. In conclusion, the comparative study of the new CSase ABC I provides two unique tools for CS/DS-related studies and applications and, more importantly, reveals the critical role of the N-terminal domain in regulating the substrate binding and degradation of these enzymes.