scholarly journals The N253F mutant structure of trehalose synthase fromDeinococcus radioduransreveals an open active-site topology

2017 ◽  
Vol 73 (11) ◽  
pp. 588-594
Author(s):  
Sih-Yao Chow ◽  
Yung-Lin Wang ◽  
Yu-Chiao Hsieh ◽  
Guan-Chiun Lee ◽  
Shwu-Huey Liaw
Keyword(s):  
Active Site ◽  
Deinococcus Radiodurans ◽  
Glycoside Hydrolase Family ◽  
Trehalose Synthase ◽  
Site Analysis ◽  
Mechanistic Analysis ◽  
Opening And Closing ◽  
Rate Limiting ◽  
Hydrolase Family ◽  
Domain Rotation

Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures ofDeinococcus radioduransTS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes.

scholarly journals Mechanistic Analysis of Trehalose Synthase from Mycobacterium smegmatis

2011 ◽  
Vol 286 (41) ◽  
pp. 35601-35609 ◽  
Author(s):  
Ran Zhang ◽  
Yuan T. Pan ◽  
Shouming He ◽  
Michael Lam ◽  
Gary D. Brayer ◽  
...  
Keyword(s):  
Isomerization Reaction ◽  
Glycoside Hydrolase Family ◽  
Hydrolytic Cleavage ◽  
Trehalose Synthase ◽  
Enzyme Active Site ◽  
Exogenous Glucose ◽  
Protease Digestion ◽  
Mechanistic Analysis ◽  
Pharmacological Interest ◽  
Opening And Closing

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of α-aryl glucosides as well as α-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp230 was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of kcat upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site.

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 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 Structures of trehalose synthase fromDeinococcus radioduransreveal that a closed conformation is involved in catalysis of the intramolecular isomerization

2014 ◽  
Vol 70 (12) ◽  
pp. 3144-3154 ◽  
Author(s):  
Yung-Lin Wang ◽  
Sih-Yao Chow ◽  
Yi-Ting Lin ◽  
Yu-Chiao Hsieh ◽  
Guan-Chiun Lee ◽  
...  
Keyword(s):  
Active Site ◽  
Deinococcus Radiodurans ◽  
Mutational Analysis ◽  
Side Reaction ◽  
Trehalose Synthase ◽  
Isomerase Activity ◽  
Closed Conformation ◽  
Dimeric Interface ◽  
Small Pore ◽  
Simple Conversion

Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant ofDeinococcus radioduranstrehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the −1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8–9-fold and an increased hydrolase activity by 1.5–1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.

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.

Study of the Active Site Residues of a Glycoside Hydrolase Family 8 Xylanase

2005 ◽  
Vol 354 (2) ◽  
pp. 425-435 ◽  
Author(s):  
T. Collins ◽  
D. De Vos ◽  
A. Hoyoux ◽  
S.N. Savvides ◽  
C. Gerday ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 8

scholarly journals Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariana A. B. Morais ◽  
Joan Coines ◽  
Mariane N. Domingues ◽  
Renan A. S. Pirolla ◽  
Celisa C. C. Tonoli ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Open Architecture ◽  
Reaction Pathways ◽  
Main Reaction ◽  
Glycoside Hydrolase Family ◽  
X Ray ◽  
Glycoside Hydrolase Family 43 ◽  
Xylanolytic Enzymes ◽  
Hydrolase Family

AbstractXylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.

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