Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from Trichoderma reesei

Zou, Jiezhong; Kleywegt, Gerard J.; Ståhlberg, Jerry; Driguez, Hugues; Nerinckx, Wim; Claeyssens, Marc; Koivula, Anu; Teeri, Tuula T.; Jones, T. Alwyn

doi:10.1016/s0969-2126(99)80171-3

Cited by 164 publications

(230 citation statements)

References 45 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: 79%

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: 79%

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

et al. 2002

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“…The density for a glucopyranosyl residue at this position is weak, which makes the 2 S O conformation less certain. However, there is no support for any other sugar conformation, and the 2 S O skew boat conformation was previously observed in wild type TfuCel6B and GH6 enzymes (23,32,67,68). Two water molecules are retained between the anomeric carbon and the proposed catalytic base, Asp-226, which adopts the "active" conformation.…”

Section: Resultsmentioning

confidence: 83%

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Vuong

et al. 2013

Journal of Biological Chemistry

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Background: Family 6 glycoside hydrolases represent an important, diverse enzyme class in cellulolytic organisms. Results: We solved structures of two Thermobifida fusca Cel6B (TfuCel6B) cellobiohydrolase mutants and examined ligand dynamics and product release with simulation. Conclusion: These results suggest mechanisms for product release in TfuCel6B. Significance: This study further elucidates the mechanism of a unique cellobiohydrolase with an extended, enclosed active site tunnel.

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“…2 and 3). These phenomena of induced fit and loop closure upon substrate binding are not uncommon in glycosidases acting on other polymeric substrates such as, for example, cellulose (28)(29)(30). It is further worth noting that although the NAG 5 substrate used for the EQ_NAG5 complex does not reach the chitin binding domain, this domain refines to somewhat different positions in the complexes described here (for instance, EQ_NAG5 vs. EQ, 6.5 o change in the angle between the chitin binding and the catalytic domains, with backbone shifts of up to 3.2 Å).…”

Section: Resultsmentioning

confidence: 95%

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

Aalten

Komander

Synstad

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

415

499

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Chitinase B (ChiB) from Serratia marcescens is a family 18 exochitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrateassisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites ؊2 to ؉3) shows closure of the ''roof'' of the active site tunnel. It also shows that the sugar in the ؊1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the ؉1 and ؉2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.C hitinases hydrolyze chitin, a linear polymer of ␤-(1,4)-linked N-acetylglucosamine (NAG), which is an abundant biopolymer. These enzymes are essential to chitin-containing organisms (fungi, insects, crustaceans) and are used by several bacteria to exploit chitin as a source of energy. Chitinase inhibitors have generated a lot of interest given their potential as insecticides (1), fungicides (2, 3), and antimalarials (4, 5). Biotechnological exploitation of chitinases, as well as design of inhibitors with sufficiently high selectivity and affinity, requires detailed knowledge of the catalytic mechanism and enzyme-substrate interactions.Most nonplant chitinases belong to glycosidase family 18 (6) and degrade chitin with retention of the stereochemistry at the anomeric carbon (7-10). The reaction is thought to be initiated by distortion of the Ϫ1 sugar ring and protonation of the glycosidic oxygen by a protonated acidic residue. The subsequent nucleophilic attack differs from classical reaction mechanisms of retaining enzymes such as lysozyme (11) and amylases (12) in that it involves the N-acetyl group of the Ϫ1 sugar, rather than a carboxylate side chain on the protein (8,9,13,14). Thus, the first step of chitinolysis results in cleavage of the sugar chain and formation of an oxazolinium ion intermediate, and hydrolysis of this ion completes the reaction (9, 15) (Fig. 1).Although the current model for the catalytic mechanism is well established, the amount of structural evidence in its support is limited (8). Important elements of the proposed mechanism were inferred from modeling studies and structures of glycosidases that do not belong to f...

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Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from Trichoderma reesei

Cited by 164 publications

References 45 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

Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

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