Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase

Sabini, Elisabetta; Sulzenbacher, G.; Dauter, Mirosława; Dauter, Zbigniew; Jørgensen, P. E. V.; Schülein, M.; Dupont, Claude; Davies, G.J.; Wilson, K.S.

doi:10.1016/s1074-5521(99)80066-0

Cited by 136 publications

(127 citation statements)

References 39 publications

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“…Recently the structure of an inactive mutant of the Xyn11 from Bacillus agaradhaerens in complex with xylotriose has been obtained (17). The interactions are very similar to that described at subsites Ϫ2 and Ϫ1 for the covalent intermediate complex (16), with the addition of the interactions for the Ϫ3 subsite.…”

Section: Production and Structural Properties Of The Xylanase Vari-mentioning

confidence: 71%

“…5, structural alignment of the above xylanases with that of A. niger enzyme showed that four of the A. niger xylanase variants (Y10A, Y89A, Y164A, and W172A) have been mutated at positions corresponding to residues that have been involved in discrete subsites of other family 11 xylanases. Tyr 10 corresponds to Trp Xyn11 , all involved in stacking interaction with the xylose ring in substite Ϫ2 of these xylanases (13,16,17,22,26 XYNII , which are thought to determine subsites ϩ1 and ϩ2 of XYNI and XYNII enzyme, respectively (22). Moreover, the oxygen atom OH of residue Tyr 6 is in the same position as atom Glu…”

Section: Production and Structural Properties Of The Xylanase Vari-mentioning

confidence: 99%

“…According to this mechanism, the catalytic residues act as a nucleophile and an acid/base catalyst, respectively (12). The three-dimensional structures of 12 family 11 endoxylanases are available from both bacteria (13)(14)(15)(16)(17)(18)(19) and fungi (20 -27). All reported structures of family 11 endoxylanases present one single (catalytic) domain with an all-␤-strand "sandwich-like" fold containing two ␤-sheets forming a large cleft that can accommodate the xylan polymers.…”

mentioning

confidence: 99%

“…The active site contains two conserved glutamate residues located on either side of the extended open cleft, which have been identified as the nucleophilic and acid/ base catalysts (28). Only a few ligand-enzyme complexes (13,16,17,29,30) have been crystallized. The subsites that bind the glycone or aglycone regions of the substrate are prefixed by Ϫ and ϩ, respectively, and the number that identifies each subsite is related to the proximity to the site of bond cleavage (31).…”

mentioning

confidence: 99%

“…The cleavage by definition takes place between subsites Ϫ1 and ϩ1. Subsites Ϫ3, Ϫ2, and Ϫ1 are well characterized by the inspection of the complex structures (13,16,17), but the characterization of the aglycone subsites is based only on modeling (22,26). Aspergillus niger xylanase is a 20-kDa family 11 xylanase with a pI and a pH optimum of 3.5 (32,33) for which an x-ray crystal structure is available (24).…”

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confidence: 99%

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Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Tahir

Berrin

Flatman

et al. 2002

Journal of Biological Chemistry

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The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k cat and K m , respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K m and decreased k cat . Tyr 6 , Tyr 10 , Tyr 89 , Tyr 164 , and Trp 172 are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the threedimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases.

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Section: Production and Structural Properties Of The Xylanase Vari-mentioning

confidence: 71%

Section: Production and Structural Properties Of The Xylanase Vari-mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Tahir

Berrin

Flatman

et al. 2002

Journal of Biological Chemistry

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Synthese konformativ fixierter Kohlenhydrate: Eine Skew-Bootkonformation derL-Iduronsäure in Heparin bestimmt dessen antithrombotische Aktivität

et al. 2001

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Die konformative Flexibilität der L‐Iduronsäure ist die Schlüsseleigenschaft dieser typischen Komponente von Heparin. Drei Pentasaccharide wurden nun synthetisiert, die Analoga des aktiven Bereichs von Heparin sind und in denen die einzelne L‐Iduronsäure in der 1C4‐, der 4C1‐ bzw. der 2S0‐Form fixiert ist. Nur die 2S0‐Variante 1 konnte die inhibierende Wirkung von Antithrombin auf den Blutkoagulationsfaktor Xa deutlich erhöhen.

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Synthesis of Conformationally Locked Carbohydrates: A Skew-Boat Conformation ofL-Iduronic Acid Governs the Antithrombotic Activity of Heparin

Das

Mallet

Esnault

et al. 2001

Angew. Chem. Int. Ed.

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Conformational flexibility of L‐iduronic acid is a key feature of this typical component of heparin. Three pentasaccharides have now been synthesized, which are analogues of the active site of heparin and in which the single L‐iduronic acid is conformationally locked in either the 1C4, 4C1, or 2S0 form. Only the 2S0 variant 1 was able to fully activate antithrombin inhibition of the blood coagulation factor Xa.

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Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase

Cited by 136 publications

References 39 publications

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Synthese konformativ fixierter Kohlenhydrate: Eine Skew-Bootkonformation derL-Iduronsäure in Heparin bestimmt dessen antithrombotische Aktivität

Synthesis of Conformationally Locked Carbohydrates: A Skew-Boat Conformation ofL-Iduronic Acid Governs the Antithrombotic Activity of Heparin

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