scholarly journals Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mounir Benkoulouche ◽  
Akli Ben Imeddourene ◽  
Louis-Antoine Barel ◽  
Guillaume Le Heiget ◽  
Sandra Pizzut ◽  
...  
Keyword(s):  
Active Site ◽  
Shigella Flexneri ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Active Enzymes ◽  
Enzyme Active Site ◽  
Molecular Determinants ◽  
Dynamics Simulations ◽  
Carbohydrate Components ◽  
Glycosylation Reaction ◽  
Hydrolase Family

AbstractThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

scholarly journals Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

2021 ◽  
Vol 11 (9) ◽  
pp. 4048
Author(s):  
Javier A. Linares-Pastén ◽  
Lilja Björk Jonsdottir ◽  
Gudmundur O. Hreggvidsson ◽  
Olafur H. Fridjonsson ◽  
Hildegard Watzlawick ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Ligand Docking ◽  
Glycoside Hydrolase Family ◽  
Ligand Interactions ◽  
Tim Barrel ◽  
Branching Point ◽  
The Difference ◽  
Dynamics Simulations ◽  
And Function ◽  
Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

Active-pocket size differentiating insectile from bacterial chitinolytic β-N-acetyl-D-hexosaminidases

2011 ◽  
Vol 438 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Tian Liu ◽  
Haitao Zhang ◽  
Fengyi Liu ◽  
Lei Chen ◽  
Xu Shen ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase Family ◽  
Complex Structures ◽  
Ostrinia Furnacalis ◽  
Wild Type ◽  
Structural Differences ◽  
Streptomyces Plicatus ◽  
Species Specific ◽  
Micro Organisms ◽  
Hydrolase Family

Chitinolytic β-N-acetyl-D-hexosaminidase is a branch of the GH20 (glycoside hydrolase family 20) β-N-acetyl-D-hexosaminidases that is only distributed in insects and micro-organisms, and is therefore a potential target for the action of insecticides. PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate] was initially identified as an inhibitor against GH20 β-N-acetyl-D-hexosaminidases. So far no crystal structure of PUGNAc in complex with any GH20 β-N-acetyl-D-hexosaminidase has been reported. We show in the present study that the sensitivities of chitinolytic β-N-acetyl-D-hexosaminidases towards PUGNAc can vary by 100-fold, with the order being OfHex1 (Ostrinia furnacalis β-N-acetyl-D-hexosaminidase)<SmCHB (Serratia marcescens chitobiase)<SpHex (Streptomyces plicatus β-N-acetyl-D-hexosaminidase). To explain this difference, the crystal structures of wild-type OfHex1 as well as mutant OfHex1(V327G) in complex with PUGNAc were determined at 2.0 Å (1 Å=0.1 nm) and 2.3 Å resolutions and aligned with the complex structures of SpHex and SmCHB. The results showed that the sensitivities of these enzymes to PUGNAc were determined by the active pocket size, with OfHex1 having the largest but narrowest entrance, whereas SpHex has the smallest entrance, suitable for holding the inhibitor, and SmCHB has the widest entrance. By widening the size of the active pocket entrance of OfHex1 through replacing the active site Val327 with a glycine residue, the sensitivity of OfHex1 to PUGNAc became similar to that of SmCHB. The structural differences among chitinolytic β-N-acetyl-D-hexosaminidases leading to different sensitivities to PUGNAc may be useful for developing species-specific pesticides and bactericides.

scholarly journals Characterization of a novel multidomain CE15-GH8 enzyme encoded by a polysaccharide utilization locus in the human gut bacterium Bacteroides eggerthii

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cathleen Kmezik ◽  
Daniel Krska ◽  
Scott Mazurkewich ◽  
Johan Larsbrink
Keyword(s):  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Active Enzymes ◽  
Human Gut ◽  
Glucuronoyl Esterase ◽  
Cob Biomass ◽  
Polysaccharide Utilization Loci ◽  
Hydrolase Family ◽  
Discrete Functions

AbstractBacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions—multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

scholarly journals Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

2015 ◽  
Vol 290 (19) ◽  
pp. 11819-11832 ◽  
Author(s):  
Christopher M. Bianchetti ◽  
Taichi E. Takasuka ◽  
Sam Deutsch ◽  
Hannah S. Udell ◽  
Eric J. Yik ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

scholarly journals Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

2000 ◽  
Vol 347 (3) ◽  
pp. 865-873 ◽  
Author(s):  
Patricia NTARIMA ◽  
Wim NERINCKX ◽  
Klaus KLARSKOV ◽  
Bart DEVREESE ◽  
Mahalingeshwara K. BHAT ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Thermomyces Lanuginosus ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
X Ray ◽  
Order Of Magnitude ◽  
Site Markers ◽  
Hydrolase Family

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

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