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602 a.a.
Key reference
DOI no: 10.1016/S0969-2126(01)00673-6 Structure 9:1005-1016 (2001) PubMed id: 11709165
Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase. M.Hrmova, J.N.Varghese, R.De Gori, B.J.Smith, H.Driguez, G.B.Fincher. ABSTRACT
BACKGROUND: Barley beta-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from beta-D-glucans and beta-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. RESULTS: When conduritol B epoxide and 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Odelta1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4(I), 4(III), 4(V)-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the -1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 A) with the Oepsilon2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C(1) conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. CONCLUSIONS: X-ray crystallography is used to define the three key intermediates during catalysis by beta-D-glucan glucohydrolase. Before a new hydrolytic event begins, the bound product (glucose) from the previous catalytic reaction is displaced by the incoming substrate, and a new enzyme-substrate complex is formed. The second stage of the hydrolytic pathway involves glycosidic bond cleavage, which proceeds through a double-displacement reaction mechanism. The crystallographic analysis of the S-cellobioside-enzyme complex with quantum mechanical modeling suggests that the complex might mimic the oxonium intermediate rather than the enzyme-substrate complex.
Selected figure(s)
Figure 4.
Figure 4. Stereo Representation of Ligands Bound in the Active Site of b Image glucan GlucohydrolaseMOLSCRIPT [47] diagrams of the nearest hydrogen bonding interactions (dashed lines) between:(a) Glucose.(b) Cyclohexitol ring.(c) 2-deoxy-2-fluoro-a- Image -glucosyl moiety.(d) S-cellobioside moiety.and the contact amino acid residues.Ligands are colored in cyan. The molecular surfaces of domains 1 and 2 are represented by transparent cyan and magenta surfaces, respectively, and are generated using GRASP [48]. Black, red, blue, yellow, and gray spheres represent carbon, oxygen, nitrogen, sulfur, and fluorine atoms, respectively. Water molecules are represented as red spheres. In (c), residues E220, E287, R291, and E491, along with Wat2 and Wat3, are not included, to improve the clarity of the data. The entrance to the active site in (b) and (c) is located perpendicularly to the page and is located toward the lower left hand corner in (a) and (d)
The above figure is reprinted by permission from Cell Press: Structure (2001, 9, 1005-1016) copyright 2001. Figure was selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
17252125 T.M.Gloster, R.Madsen, and G.J.Davies (2007).
Structural basis for cyclophellitol inhibition of a beta-glucosidase.Org Biomol Chem, 5, 444-446.
PDB code: 2jal
16717412 H.Li, G.Zhao, H.Miyake, H.Umekawa, T.Kimura, K.Ohmiya, and K.Sakka (2006).
Identification of a catalytic residue of Clostridium paraputrificum N-acetyl-beta-D-glucosaminidase Nag3A by site-directed mutagenesis.Biosci Biotechnol Biochem, 70, 1127-1133.
15853815 J.Jänis, J.Hakanpää, N.Hakulinen, F.M.Ibatullin, A.Hoxha, P.J.Derrick, J.Rouvinen, and P.Vainiotalo (2005).
Determination of thioxylo-oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X-ray crystallography.FEBS J, 272, 2317-2333.
PDB code: 1xnk
15817452 L.Premkumar, A.R.Sawkar, S.Boldin-Adamsky, L.Toker, I.Silman, J.W.Kelly, A.H.Futerman, and J.L.Sussman (2005).
X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease.J Biol Chem, 280, 23815-23819.
PDB code: 1y7v
15170117 L.Ying, M.Kitaoka, and K.Hayashi (2004).
Effects of truncation at the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens.Biosci Biotechnol Biochem, 68, 1113-1118.
14597633 M.Hrmova, R.De Gori, B.J.Smith, A.Vasella, J.N.Varghese, and G.B.Fincher (2004).
Three-dimensional structure of the barley beta-D-glucan glucohydrolase in complex with a transition state mimic.J Biol Chem, 279, 4970-4980.
PDB code: 1lq2
12595701 A.Varrot, and G.J.Davies (2003).
Direct experimental observation of the hydrogen-bonding network of a glycosidase along its reaction coordinate revealed by atomic resolution analyses of endoglucanase Cel5A.Acta Crystallogr D Biol Crystallogr, 59, 447-452.
PDB codes: 1h11 1h2j 1hf6
12464603 R.C.Lee, M.Hrmova, R.A.Burton, J.Lahnstein, and G.B.Fincher (2003).
Bifunctional family 3 glycoside hydrolases from barley with alpha -L-arabinofuranosidase and beta -D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing.J Biol Chem, 278, 5377-5387. The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.
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