Snapshots along an Enzymatic Reaction Coordinate:  Analysis of a Retaining β-Glycoside Hydrolase<sup>,</sup>

Davies, G.J.; Mackenzie, Lloyd; Varrot, A.; Dauter, Mirosława; Brzozowski, A.M.; Schülein, M.; Withers, Stephen G.

doi:10.1021/bi981315i

Cited by 264 publications

(266 citation statements)

References 41 publications

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“…This is in contrast to several well-characterized exo-and endoacting ␤-D-glycoside hydrolases in which the subsite Ϫ1 glycosyl residues are distorted significantly (Sulzenbacher et al, 1996;Tews et al, 1996Tews et al, , 1997Davies et al, 1998;Zou et al, 1999;Fort et al, 2001;Papanikolau et al, 2001). …”

Section: Discussioncontrasting

confidence: 80%

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

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Family 3 ␤ -D -glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of ␤ -D -glucosyl residues from nonreducing termini of a range of ␤ -D -glucans and ␤ -D -oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley ␤ -D -glucan glucohydrolase in complex with nonhydrolyzable S -glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite ؊ 1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite ؉ 1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite ؉ 1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent ␤ -D -glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development. INTRODUCTION␤ -D -Glucan glucohydrolases have been purified and characterized from barley seedlings, maize coleoptiles, soybean cultures, Acacia cells, nasturtium cells, and cultured tobacco cells (Cline and Albersheim, 1981;Nari et al., 1982;Lienart et al., 1986;Labrador and Nevins, 1989;Hrmova et al., 1996;Kotake et al., 1997;Crombie et al., 1998;Kim et al., 2000;Koizumi et al., 2000). They can hydrolyze glycosidic linkages in several ␤ -D -glucans, in ␤ -D -oligoglucosides containing (1 → 2)-, (1 → 3)-, (1 → 4)-, or (1 → 6)-linkages, in aryl ␤ -D -glucosides such as 4 Ј -nitrophenyl ␤ -D -glucopyranoside (4NPGlc), and in some ␤ -D -oligoxyloglucosides (Crombie et al., 1998;Hrmova and Fincher, 1998;Kim et al., 2000). The barley ␤ -D -glucan glucohydrolases also hydrolyze cyanogenic ␤ -D -glucosides, albeit with low activity (M. Hrmova and G.B. Fincher, unpublished data). Single Glc molecules are released from the nonreducing termini of these substrates, with retention of the anomeric configuration (Hrmova et al., 1996). Their broad substrate specificity makes it difficult to assign these higher plant ␤ -D -glucan glucohydrolases to current Enzyme Commission classes; therefore, they have been described variously as ␤ -D -glucan glucohydrolases, (1 → 3)-␤ -D -glucan exohydrolases, and ␤ -D -glucosidases. Nevertheless, they can be classified according to the structural criteria of Henrissat (1991) and fall into the family 3 group of glycoside hydrolases (http://afmb.cnrsmrs.fr/CAZY/).The distribution of the broad-specificity ␤ -D -glucan glucohydrolases in various tissues of monocotyledons and dicotyledons, together with the presence of expressed sequence tags in gymnosperm sequence databases (e.g., Pinus taeda ), suggests that they may play a fundamental role in plant growth and development...

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Section: Discussioncontrasting

confidence: 80%

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

et al. 2002

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“…Secondly, there is no sign of distortion towards the half chair of the transition state such as that observed for the bound substrate. Undistorted intermediates have also been seen in several b-retaining enzymes 2,7 . The lack of distortion indicates that the catalytic subsite -1 is stereochemically complementary to the intermediate, similar to a situation also seen in, for example, the sulfur transferase rhodanese 23 .…”

Section: How Cgtase Binds Its Substratementioning

confidence: 92%

“…3 Stereoview of the covalent intermediate electron density from a 2F o -F c omit 37 map (1s contoured), the arrow indicates the glycosyl-enzyme covalent bond. In contrast to earlier work with other enzymes, neither the nucleophilic amino acid nor the glucose at the catalytic subsite -1 has been chemically modified 2,7 . Comparing the intermediate ring puckering parameters with those of small molecule X-ray structures and a potential map from molecular mechanics calculations indicates that the sugar bound to Asp 229 has a low-energy 4 C 1 chair conformation 15 .…”

Section: Mechanism Of the First Reaction Stepmentioning

confidence: 99%

“…Glycosyl hydrolases and transferases employ four basic mechanisms, named according to the anomeric configuration of the substrate (a or b-glycosidic bonds) and the stereochemical outcome of the reaction (retention or inversion) 4 . The b-retaining mechanism of hen egg white lysozyme is a wellknown example 6,7 . The a-retaining mechanism is used by enzymes from the a-amylase family (glycosyl hydrolase family 13; ref.…”

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confidence: 99%

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et al. 1999

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“…Such a conformation does not resemble the predicted transition state geometry but rather mimics those observed for the Michaelis complexes of retaining enzymes active on b-glycosides of the glucoseries (for example, ref. 35,36) and reflects in-line geometry for nucleophilic substitution at the anomeric centre: the O-C2-N2 (O-C1-N1) angle is 167 • . Such distortion appears critical for establishing a powerful electrostatic interaction between the amine 7 and Asp 243.…”

Section: Resultsmentioning

confidence: 99%

A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

et al. 2007

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The synthesis of an analogue of 6-epi-valienamine bearing an acetamido group and its characterisation as an inhibitor of b-N-acetylglucosaminidases are described. The compound is a good inhibitor of both human O-GlcNAcase and human b-hexosaminidase, as well as two bacterial b-N-acetylglucosaminidases. A 3-D structure of the complex of Bacteroides thetaiotaomicron BtGH84 with the inhibitor shows the unsaturated ring is surprisingly distorted away from its favoured solution phase conformation and reveals potential for improved inhibitor potency.

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Snapshots along an Enzymatic Reaction Coordinate: Analysis of a Retaining β-Glycoside Hydrolase^,

Cited by 264 publications

References 41 publications

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

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A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

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Snapshots along an Enzymatic Reaction Coordinate: Analysis of a Retaining β-Glycoside Hydrolase,

Cited by 264 publications

References 41 publications

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

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A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

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Snapshots along an Enzymatic Reaction Coordinate: Analysis of a Retaining β-Glycoside Hydrolase^,