Carbohydrate‐binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

AbstractChemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a ‘non-catalytic’ lectin-like or carbohydrate-binding domains (CBMs) in the catalytic step for glycosidic bond synthesis using β-cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. Our study provides evidence for the direct participation of CBMs in the catalytic reaction step for β-glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intra-domain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM-based CAZyme chimeras for synthesis of bespoke oligosaccharides using simple activated sugar monomers.

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Glucoamylase starch-binding domain of Aspergillus niger B1: molecular cloning and functional characterization

Biochemical Journal ◽

10.1042/bj20021527 ◽

2003 ◽

Vol 372 (3) ◽

pp. 905-910 ◽

Cited By ~ 18

Author(s):

Tzur PALDI ◽

Ilan LEVY ◽

Oded SHOSEYOV

Keyword(s):

Aspergillus Niger ◽

Corn Starch ◽

Catalytic Domain ◽

Functional Characterization ◽

Carbohydrate Binding ◽

Amylolytic Enzymes ◽

Binding Domains ◽

C Terminus ◽

Carbohydrate Binding Modules ◽

Modified Starches

Carbohydrate-binding modules (CBMs) are protein domains located within a carbohydrate-active enzyme, with a discrete fold that can be separated from the catalytic domain. Starch-binding domains (SBDs) are CBMs that are usually found at the C-terminus in many amylolytic enzymes. The SBD from Aspergillus niger B1 (CMI CC 324262) was cloned and expressed in Escherichia coli as an independent domain and the recombinant protein was purified on starch. The A. niger B1 SBD was found to be similar to SBD from A. kawachii, A. niger var. awamori and A. shirusami (95–96% identity) and was classified as a member of the CBM family 20. Characterization of SBD binding to starch indicated that it is essentially irreversible and that its affinity to cationic or anionic starch, as well as to potato or corn starch, does not differ significantly. These observations indicate that the fundamental binding area on these starches is essentially the same. Natural and chemically modified starches are among the most useful biopolymers employed in the industry. Our study demonstrates that SBD binds effectively to both anionic and cationic starch.

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Structural insight into industrially relevant glucoamylases: flexible positions of starch-binding domains

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318004989 ◽

2018 ◽

Vol 74 (5) ◽

pp. 463-470 ◽

Cited By ~ 3

Author(s):

Christian Roth ◽

Olga V. Moroz ◽

Antonio Ariza ◽

Lars K. Skov ◽

Keiichi Ayabe ◽

...

Keyword(s):

Catalytic Domain ◽

Relative Concentration ◽

Spatial Arrangement ◽

Carbohydrate Binding ◽

Polymeric Substrate ◽

Binding Domains ◽

Catalytic Mechanisms ◽

The Individual ◽

First Time ◽

Insight Into

Glucoamylases are one of the most important classes of enzymes in the industrial degradation of starch biomass. They consist of a catalytic domain and a carbohydrate-binding domain (CBM), with the latter being important for the interaction with the polymeric substrate. Whereas the catalytic mechanisms and structures of the individual domains are well known, the spatial arrangement of the domains with respect to each other and its influence on activity are not fully understood. Here, the structures of three industrially used fungal glucoamylases, two of which are full length, have been crystallized and determined. It is shown for the first time that the relative orientation between the CBM and the catalytic domain is flexible, as they can adopt different orientations independently of ligand binding, suggesting a role as an anchor to increase the contact time and the relative concentration of substrate near the active site. The flexibility in the orientations of the two domains presented a considerable challenge for the crystallization of the enzymes.

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Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

Journal of Bacteriology ◽

10.1128/jb.181.15.4611-4616.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4611-4616 ◽

Cited By ~ 29

Author(s):

Helen D. Simpson ◽

Frederic Barras

Keyword(s):

Escherichia Coli ◽

Catalytic Domain ◽

Three Dimensional ◽

Site Directed Mutagenesis ◽

Erwinia Chrysanthemi ◽

Dimensional Structure ◽

Carbohydrate Binding ◽

Binding Domains ◽

Cellulose Binding

ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

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The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16014072 ◽

2016 ◽

Vol 72 (10) ◽

pp. 750-761 ◽

Cited By ~ 4

Author(s):

Emma L. Summers ◽

Christina D. Moon ◽

Renee Atua ◽

Vickery L. Arcus

Keyword(s):

Glycoside Hydrolase ◽

Catalytic Domain ◽

Binding Domain ◽

Carbohydrate Binding ◽

Catalytic Core ◽

Binding Domains ◽

Carbohydrate Binding Modules ◽

Tim Barrel ◽

Domain Arrangement ◽

Hydrolysis Of

Glycoside hydrolase (GH) family 29 consists solely of α-L-fucosidases. These enzymes catalyse the hydrolysis of glycosidic bonds. Here, the structure of GH29_0940, a protein cloned from metagenomic DNA from the rumen of a cow, has been solved, which reveals a multi-domain arrangement that has only recently been identified in bacterial GH29 enzymes. The microbial species that provided the source of this enzyme is unknown. This enzyme contains a second carbohydrate-binding domain at its C-terminal end in addition to the typical N-terminal catalytic domain and carbohydrate-binding domain arrangement of GH29-family proteins. GH29_0940 is a monomer and its overall structure consists of an N-terminal TIM-barrel-like domain, a central β-sandwich domain and a C-terminal β-sandwich domain. The TIM-barrel-like catalytic domain exhibits a (β/α)8/7arrangement in the core instead of the typical (β/α)8topology, with the `missing' α-helix replaced by a long meandering loop that `closes' the barrel structure and suggests a high degree of structural flexibility in the catalytic core. This feature was also noted in all six other structures of GH29 enzymes that have been deposited in the PDB. Based on sequence and structural similarity, the residues Asp162 and Glu220 are proposed to serve as the catalytic nucleophile and the proton donor, respectively. Like other GH29 enzymes, the GH29_0940 structure shows five strictly conserved residues in the catalytic pocket. The structure shows two glycerol molecules in the active site, which have also been observed in other GH29 structures, suggesting that the enzyme catalyses the hydrolysis of small carbohydrates. The two binding domains are classed as family 32 carbohydrate-binding modules (CBM32). These domains have residues involved in ligand binding in the loop regions at the edge of the β-sandwich. The predicted substrate-binding residues differ between the modules, suggesting that different modules bind to different groups on the substrate(s). Enzymes that possess multiple copies of CBMs are thought to have a complex mechanism of ligand recognition. Defined electron density identifying a long 20-amino-acid hydrophilic loop separating the two CBMs was observed. This suggests that the additional C-terminal domain may have a dynamic range of movement enabled by the loop, allowing a unique mode of action for a GH29 enzyme that has not been identified previously.

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The K1K2 Region of Lys-Gingipain of Porphyromonas gingivalis Blocks Induction of HLA Expression by Gamma Interferon

Infection and Immunity ◽

10.1128/iai.00528-12 ◽

2012 ◽

Vol 80 (10) ◽

pp. 3733-3741 ◽

Cited By ~ 1

Author(s):

Peter L. Yun ◽

Nan Li ◽

Charles A. Collyer ◽

Neil Hunter

Keyword(s):

Porphyromonas Gingivalis ◽

Catalytic Domain ◽

Umbilical Vein ◽

Gamma Interferon ◽

Recent Analysis ◽

Carbohydrate Binding ◽

Content Type ◽

Binding Domains ◽

Ifn Γ ◽

Umbilical Vein Endothelial Cells

ABSTRACTIn the context of periodontal disease, cysteine proteinases or gingipains fromPorphyromonas gingivalishave been implicated in the hydrolysis of cytokines, including gamma interferon (IFN-γ). This cytokine plays a crucial role in host defenses, in part, by regulating expression of major histocompatibility complex molecules. Our recent analysis has identified three structurally defined modules, K1, K2, and K3, of the hemagglutinin region of the lysine gingipain Kgp. These three structurally homologous domains have a common β-sandwich topology that is similar to that found in a superfamily of adhesins and carbohydrate binding domains. The three Kgp hemagglutinin modules are distinguished by variation in some of the loops projecting from the β-sandwich core. Recombinant products corresponding to both single and multidomain regions as well as native Kgp bound IFN-γ with similar affinities. Among the adhesin domain constructs, only the K1K2 polypeptide inhibited the upregulation of HLA-1 expression in a human erythroleukemia (K562) line induced by both recombinant and native IFN-γ. The K1K2 polypeptide also inhibited HLA-DR expression induced by IFN-γ in human umbilical vein endothelial cells. These effects were competitively inhibited by coincubation with sodium or potassium chloride solution. The N-terminal residues of IFN-γ were implicated in mediating the effect of K1K2, while antibody binding to loop 1 of K2 blocked the action of K1K2. The findings indicate the potential significance of structurally defined Kgp adhesin modules in the inactivation of IFN-γ but also the potential of K1K2 in locating the target for the catalytic domain of Kgp.

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Genomic analysis of C-type lectins

Biochemical Society Symposium ◽

10.1042/bss0690059 ◽

2002 ◽

Vol 69 ◽

pp. 59-72 ◽

Cited By ~ 85

Author(s):

Kurt Drickamer ◽

Andrew J. Fadden

Keyword(s):

Biological Effects ◽

Cell Signalling ◽

Genomic Analysis ◽

Binding Activity ◽

Immunoglobulin Superfamily ◽

Carbohydrate Binding ◽

Carbohydrate Recognition ◽

Calcium Dependent ◽

Binding Domains ◽

Carbohydrate Recognition Domains

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell–cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.

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Aromatic interactions in Glycochemistry: from molecular recognition to catalysis

Current Medicinal Chemistry ◽

10.2174/0929867328666210709120216 ◽

2021 ◽

Vol 28 ◽

Author(s):

Andrés González Santana ◽

Laura Díaz-Casado ◽

Laura Montalvillo ◽

Ester Jiménez-Moreno ◽

Enrique Mann ◽

...

Keyword(s):

Molecular Recognition ◽

Transition States ◽

Carbohydrate Binding ◽

Potential Influence ◽

Chemical Structures ◽

Receptor Complexes ◽

Binding Domains ◽

Aromatic Interactions ◽

Aromatic Complexes ◽

Recognition Motifs

: Aromatic platforms are ubiquitous recognition motifs occurring in protein carbohydrate binding domains (CBDs), RNA receptors and enzymes. They stabilize the glycoside/receptor complexes by participating in stacking CH/ interactions with either the - or - face of the corresponding pyranose units. In addition, the role played by aromatic units in the stabilization of glycoside cationic transition states has started being recognized in recent years. Extensive studies carried out during the last decade have allowed to dissect the main contributing forces that stabilize the carbohydrate/aromatic complexes, while helping delineate not only the standing relationship between the glycoside/aromatic chemical structures and the strength of this interaction, but also their potential influence on glycoside reactivity.

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Chitinase Chit62J4 Essential for Chitin Processing by Human Microbiome Bacterium Clostridium paraputrificum J4

Molecules ◽

10.3390/molecules26195978 ◽

2021 ◽

Vol 26 (19) ◽

pp. 5978

Author(s):

Jan Dohnálek ◽

Jarmila Dušková ◽

Galina Tishchenko ◽

Petr Kolenko ◽

Tereza Skálová ◽

...

Keyword(s):

Catalytic Domain ◽

Glycosyl Hydrolase ◽

Bacterial Genome ◽

Human Microbiome ◽

Culture Fluid ◽

Carbohydrate Binding ◽

Ph Optimum ◽

Catalytic Sites ◽

Tim Barrel ◽

Clostridium Paraputrificum

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s−1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains—2x Fn3/Big3 and a carbohydrate binding module—that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.

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The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

Biochemical Journal ◽

10.1042/bj3420473 ◽

1999 ◽

Vol 342 (2) ◽

pp. 473-480 ◽

Cited By ~ 27

Author(s):

Jaitinder GILL ◽

Jane E. RIXON ◽

David N. BOLAM ◽

Simon MCQUEEN-MASON ◽

Peter J. SIMPSON ◽

...

Keyword(s):

Microcrystalline Cellulose ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Wall Material ◽

Common Mechanism ◽

Type Ii ◽

Binding Domains ◽

Covalently Linked ◽

Cellulose Binding ◽

Internal Domain

Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10ACD) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBDX) or fused to the catalytic domain (Pf Xyn10A′), bound to amorphous and bacterial microcrystalline cellulose with a Ka of 2.5×105 M-1. CBDX exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A′ was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10ACD; however, Pf Xyn10A′ and Pf Xyn10ACD exhibited the same activity against soluble substrates. CBDX did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10ACD when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A′, and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10ACD via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBDX is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase.

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