Mechanism-based Labeling Defines the Free Energy Change for Formation of the Covalent Glycosyl-enzyme Intermediate in a Xyloglucan endo-Transglycosylase

Piens, Kathleen; Fauré, Régis; Sundqvist, Gustav; Baumann, Martin; Saura-Valls, Marc; Teeri, Tuula T.; Cottaz, Sylvain; Planas, Antoni; Driguez, Hugues; Brumer, Harry

doi:10.1074/jbc.m803057200

Cited by 18 publications

(19 citation statements)

References 41 publications

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“…However, the situation has been changed after the report of a crystal structure of HEWL covalently bound to C1 carbon of the Ϫ1 sugar, which exhibits a chair conformation with the C1 carbon in sp 3 hybridization (12). Nowadays, the catalytic mechanism through the covalently bound intermediate is more widely accepted by enzyme researchers (33)(34)(35). However, the crystal structure capturing the covalent glycosyl-HEWL intermediate was obtained only by use of the inactive mutant HEWL(E35Q) and 2-acetamido-2-deoxy-␤-D-glucopyranosyl-(134)-2-deoxy-2-fluoro-␤-D-glucopyranosyl fluoride (NAG2FGlcF).…”

Section: Discussionmentioning

confidence: 99%

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

Ogata

Umemoto

Ohnuma

et al. 2013

Journal of Biological Chemistry

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Backgroud: A pure and stable transition-state analogue for lysozyme has not been reported thus far. Results: We synthesized 4-O-␤-tri-N-acetyl-chitotriosyl moranoline (3), which inhibited strongly the lysozyme reaction. Conclusion: Compound 3 was found to be a novel and stable transition-state analogue for lysozyme. Significance: The crystal structure of lysozyme in a complex with 3 supports the covalent glycosyl-enzyme intermediate in the catalytic reaction. Lysozymes (EC 3.2.1.17) are glycosidases acting in the innate-immune system of most animals (1). The common feature of lysozymes is to exert antibacterial activity by cleaving the ␤-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (GlcNAc) 5 in peptidoglycan, a major bacterial cell-wall component (2). Hen egg white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by x-ray crystallography, revealing that the enzyme is divided into two domains by a deep substratebinding cleft containing the catalytic site (3). The catalytic importance of the conserved residues, Glu-35 and Asp-52, was also confirmed by the crystal structure of the enzyme-ligand complex (4) and site-directed mutagenesis (5). These studies on the structure and function of HEWL offer the strongest support available to date for the theory that enzymes may catalyze reactions by binding substrates in the geometry of the transition state more strongly than in that of the ground state. 4-O-␤-HEWL has six subsites for sugar residue binding, termed Ϫ4 to ϩ2 (formerly A, B, C, D, E, and F (6)). The enzyme cleave a glycosidic linkage between the sugar residues at subsites Ϫ1 and ϩ1 with the aid of the proton-donating action of Glu-35 as a general acid. The substrate analog tri-N-acetylchitotriose, (GlcNAc) 3 , binds predominantly to subsites Ϫ4, Ϫ3, and Ϫ2 of HEWL, keeping away from subsite Ϫ1 due to the unfavorable positive free energy of the sugar residue binding at that subsite (7). This situation might have been an obstacle to understanding the catalytic mechanism of the enzyme. In the classical explanation, the lysozyme-catalyzed reaction was recognized to take place via a carbenium ion

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Section: Discussionmentioning

confidence: 99%

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

Ogata

Umemoto

Ohnuma

et al. 2013

Journal of Biological Chemistry

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“…The stability of the PttXET16-34 glycosyl-enzyme to hydrolysis in dilute aqueous solution (56 M water) is indeed remarkable: kinetic trapping studies placed the hydrolytic half-life of this intermediate at approximately 3 h (k hydr = 0.005 min 21 ) in the complete absence of a xyloglucan acceptor substrate (which could not occur in vivo), while the addition of xyloglucan oligosaccharides rapidly turned over the glycosyl enzyme (in less than 2 min; Piens et al, 2008). This behavior stands in stark contrast to the measured hydrolytic rate of TmNXG1 on tamarind (Tamarindus indica) xyloglucan (apparent k cat = 5.5 min 21 ), whose glycosyl-enzyme is thus rapidly broken down by water (Baumann et al, 2007).…”

Section: Differences In Loop Structures Lining the Active-site Cleft mentioning

confidence: 99%

“…the resynthesis of xyloglucans from artificial donor substrates [a-fluoroglycosides of xyloglucan oligosaccharides]; Piens et al, 2007;Gullfot et al, 2009). Furthermore, mutation of Glu-85 in PttXET16-34 to Ala prevents formation of the covalent glycosyl-enzyme (Piens et al, 2008). The hydrophobic side chains of the intervening residues in the catalytic motif do not play a direct role in catalysis, as these point into the interior of the b-jellyroll structure, where they fulfill a structural function.…”

Section: The Catalytic Amino Acids: Identification and Rolesmentioning

confidence: 99%

“…3) is the formation of a covalent glycosyl-enzyme intermediate, which can be broken down by water, yielding hydrolysis (XEH activity), or an incoming saccharide substrate, yielding transglycosylation (XET activity; Planas, 2000;Gilbert et al, 2008). Definitive proof of the covalent nature of the glycosyl-enzyme has been obtained through kinetic trapping and direct observation of this intermediate in PttXET16-34 by mass spectrometry (Piens et al, 2008).…”

Section: The Catalytic Amino Acids: Identification and Rolesmentioning

confidence: 99%

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The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling

2010

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“…In the deglycosylation step, water or another nucleophile displaces the enzyme from the sugar with basic assistance from the catalytic acid/base. Transglycosylation can occur by the same mechanism in GH and TG (11). In principle, retaining GT could use this mechanism, but it was recently demonstrated that trehalose phosphate synthase uses a front end attack S N itype mechanism instead (12).…”

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confidence: 99%

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Luang

Cho

Mahong

et al. 2013

Journal of Biological Chemistry

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Background: Glycosylation regulates the activities of plant metabolites and is mediated by glycosyltransferases (GT), glycoside hydrolases (GH), and transglycosidases (TG). Results: The vacuolar TG Os9BGlu31 transfers glucose between phenolic acid esters and other compounds. Conclusion: Os9BGlu31 equilibrates phenolic acids, phytohormones, and their glucosyl conjugates. Significance: Os9BGlu31 and similar TG can broaden glycoconjugate diversity in planta.

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Mechanism-based Labeling Defines the Free Energy Change for Formation of the Covalent Glycosyl-enzyme Intermediate in a Xyloglucan endo-Transglycosylase

Cited by 18 publications

References 41 publications

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline, Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate*

The XTH Gene Family: An Update on Enzyme Structure, Function, and Phylogeny in Xyloglucan Remodeling

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

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