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PDBsum entry 1ojk

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protein ligands Protein-protein interface(s) links
Hydrolase PDB id
1ojk
Jmol PyMol
Contents
Protein chains
399 a.a. *
Ligands
GLC-BGC ×2
BGC-BGC ×2
NAG ×2
GOL ×2
Waters ×793
* Residue conservation analysis
PDB id:
1ojk
Name: Hydrolase
Title: Anatomy of glycosynthesis: structure and kinetics of the humicola insolens cel7be197a and e197s glycosynthase mutants
Structure: Endoglucanase i. Chain: a, b. Synonym: endo-1,4-beta-glucanase. Engineered: yes. Mutation: yes
Source: Humicola insolens. Organism_taxid: 34413. Expressed in: aspergillus oryzae. Expression_system_taxid: 5062
Resolution:
1.5Å     R-factor:   0.143     R-free:   0.173
Authors: V.M.-A.Ducros,C.A.Tarling,D.L.Zechel,A.M.Brzozowski, T.P.Frandsen,I.Von Ossowski,M.Schulein,S.G.Withers, G.J.Davies
Key ref:
V.M.Ducros et al. (2003). Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants. Chem Biol, 10, 619-628. PubMed id: 12890535 DOI: 10.1016/S1074-5521(03)00143-1
Date:
10-Jul-03     Release date:   07-Jan-04    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P56680  (GUN1_HUMIN) -  Endoglucanase 1
Seq:
Struc:
402 a.a.
399 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.2.1.4  - Cellulase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biological process     metabolic process   4 terms 
  Biochemical function     hydrolase activity     4 terms  

 

 
DOI no: 10.1016/S1074-5521(03)00143-1 Chem Biol 10:619-628 (2003)
PubMed id: 12890535  
 
 
Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants.
V.M.Ducros, C.A.Tarling, D.L.Zechel, A.M.Brzozowski, T.P.Frandsen, I.von Ossowski, M.Schülein, S.G.Withers, G.J.Davies.
 
  ABSTRACT  
 
The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Enzyme-Catalyzed Glycosidic Bond Formation(A) The transglycosylation reaction catalyzed by retaining glycosidases in which a covalent glycosyl-enzyme intermediate is intercepted by the acceptor alcohol (in competition with water); and (B) the reaction catalyzed by “glycosynthases” whose enzyme-derived nucleophilic carboxylate has been mutated to glycine, serine, or alanine.
Figure 2.
Figure 2. 3-D Structure of the Cellobiose Complex of the Humicola insolens Cel7B (E197S) GlycosynthaseThe protein topology is shown (helices, red; sheets, blue; coil, yellow) together with the molecular surface (wheat). Donor (−2, −1) sugars are shown in gray, and acceptor (+1, +2) in yellow “licorice”. The figure is in divergent (“wall-eyed”) stereo.
 
  The above figures are reprinted by permission from Cell Press: Chem Biol (2003, 10, 619-628) copyright 2003.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20352422 D.B.Jordan, and K.Wagschal (2010).
Properties and applications of microbial beta-D-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium.
  Appl Microbiol Biotechnol, 86, 1647-1658.  
19819900 M.Hidaka, S.Fushinobu, Y.Honda, T.Wakagi, H.Shoun, and M.Kitaoka (2010).
Structural explanation for the acquisition of glycosynthase activity.
  J Biochem, 147, 237-244.
PDB codes: 2dro 2drq 2drr 2drs 3a3v
19875083 B.Cobucci-Ponzano, F.Conte, E.Bedini, M.M.Corsaro, M.Parrilli, G.Sulzenbacher, A.Lipski, F.Dal Piaz, L.Lepore, M.Rossi, and M.Moracci (2009).
beta-Glycosyl azides as substrates for alpha-glycosynthases: preparation of efficient alpha-L-fucosynthases.
  Chem Biol, 16, 1097-1108.
PDB code: 2wsp
19543714 H.Schagerlöf, C.Nilsson, L.Gorton, F.Tjerneld, H.Stålbrand, and A.Cohen (2009).
Use of 18O water and ESI-MS detection in subsite characterisation and investigation of the hydrolytic action of an endoglucanase.
  Anal Bioanal Chem, 394, 1977-1984.  
18378673 A.M.Cartwright, E.K.Lim, C.Kleanthous, and D.J.Bowles (2008).
A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities.
  J Biol Chem, 283, 15724-15731.  
18635010 A.Sadeghi-Khomami, M.D.Lumsden, and D.L.Jakeman (2008).
Glycosidase inhibition by macrolide antibiotics elucidated by STD-NMR spectroscopy.
  Chem Biol, 15, 739-749.  
18330853 H.Tao, P.Peralta-Yahya, J.Decatur, and V.W.Cornish (2008).
Characterization of a new glycosynthase cloned by using chemical complementation.
  Chembiochem, 9, 681-684.  
19053460 P.Peralta-Yahya, B.T.Carter, H.Lin, H.Tao, and V.W.Cornish (2008).
High-throughput selection for cellulase catalysts using chemical complementation.
  J Am Chem Soc, 130, 17446-17452.  
17955483 A.Ben-David, T.Bravman, Y.S.Balazs, M.Czjzek, D.Schomburg, G.Shoham, and Y.Shoham (2007).
Glycosynthase activity of Geobacillus stearothermophilus GH52 beta-xylosidase: efficient synthesis of xylooligosaccharides from alpha-D-xylopyranosyl fluoride through a conjugated reaction.
  Chembiochem, 8, 2145-2151.  
18043802 K.Piens, A.M.Henriksson, F.Gullfot, M.Lopez, R.Fauré, F.M.Ibatullin, T.T.Teeri, H.Driguez, and H.Brumer (2007).
Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants.
  Org Biomol Chem, 5, 3971-3978.  
17376777 T.M.Gloster, F.M.Ibatullin, K.Macauley, J.M.Eklöf, S.Roberts, J.P.Turkenburg, M.E.Bjørnvad, P.L.Jørgensen, S.Danielsen, K.S.Johansen, T.V.Borchert, K.S.Wilson, H.Brumer, and G.J.Davies (2007).
Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12.
  J Biol Chem, 282, 19177-19189.
PDB codes: 2jem 2jen 2jep 2jeq
16741587 A.S.Rowan, and C.J.Hamilton (2006).
Recent developments in preparative enzymatic syntheses of carbohydrates.
  Nat Prod Rep, 23, 412-443.  
16717424 M.Sugimura, M.Nishimoto, and M.Kitaoka (2006).
Characterization of glycosynthase mutants derived from glycoside hydrolase family 10 xylanases.
  Biosci Biotechnol Biochem, 70, 1210-1217.  
16301312 Y.Honda, and M.Kitaoka (2006).
The first glycosynthase derived from an inverting glycoside hydrolase.
  J Biol Chem, 281, 1426-1431.  
16688347 Y.W.Kim, D.T.Fox, O.Hekmat, T.Kantner, L.P.McIntosh, R.A.Warren, and S.G.Withers (2006).
Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi.
  Org Biomol Chem, 4, 2025-2032.  
15866000 K.L.Morley, and R.J.Kazlauskas (2005).
Improving enzyme properties: when are closer mutations better?
  Trends Biotechnol, 23, 231-237.  
15062769 A.M.Daines, B.A.Maltman, and S.L.Flitsch (2004).
Synthesis and modifications of carbohydrates, using biotransformations.
  Curr Opin Chem Biol, 8, 106-113.  
15062085 J.Allouch, W.Helbert, B.Henrissat, and M.Czjzek (2004).
Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose.
  Structure, 12, 623-632.
PDB code: 1urx
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 codes are shown on the right.

 

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