4AX6

HYPOCREA JECORINA CEL6A D221A MUTANT SOAKED WITH 6-CHLORO-4- PHENYLUMBELLIFERYL-BETA-CELLOBIOSIDE


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.30 Å
  • R-Value Free: 0.254 
  • R-Value Work: 0.204 
  • R-Value Observed: 0.206 

Starting Model: experimental
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This is version 2.2 of the entry. See complete history


Literature

Rational Design, Synthesis, Evaluation and Enzyme-Substrate Structures of Improved Fluorogenic Substrates for Family 6 Glycoside Hydrolases.

Wu, M.Nerinckx, W.Piens, K.Ishida, T.Hansson, H.Sandgren, M.Stahlberg, J.

(2013) FEBS J 280: 184

  • DOI: https://doi.org/10.1111/febs.12060
  • Primary Citation of Related Structures:  
    4AU0, 4AX6, 4AX7

  • PubMed Abstract: 

    Methylumbelliferyl-β-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain β-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF-G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-β-cellobiosides [6-chloro-4-methyl- (ClMUF); 6-chloro-4-trifluoromethyl- (ClF3MUF); 4-phenyl- (PhUF); 6-chloro-4-phenyl- (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10-150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of k(cat) and K(M) for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF- and ClPhUF-β-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF-G2.


  • Organizational Affiliation

    Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
EXOGLUCANASE 2
A, B
363Trichoderma reeseiMutation(s): 1 
EC: 3.2.1.91
UniProt
Find proteins for P07987 (Hypocrea jecorina)
Explore P07987 
Go to UniProtKB:  P07987
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP07987
Glycosylation
Glycosylation Sites: 9
Sequence Annotations
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  • Reference Sequence
Oligosaccharides

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Entity ID: 2
MoleculeChains Length2D Diagram Glycosylation3D Interactions
beta-D-glucopyranose-(1-4)-beta-D-glucopyranose
C, D
2N/A
Glycosylation Resources
GlyTouCan:  G84824ZO
GlyCosmos:  G84824ZO
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
UWU
Query on UWU

Download Ideal Coordinates CCD File 
N [auth A],
V [auth B]
6-chloranyl-7-oxidanyl-4-phenyl-chromen-2-one
C15 H9 Cl O3
TUQNFQUMZKUALD-UHFFFAOYSA-N
NAG
Query on NAG

Download Ideal Coordinates CCD File 
E [auth A],
F [auth A],
O [auth B],
P [auth B]
2-acetamido-2-deoxy-beta-D-glucopyranose
C8 H15 N O6
OVRNDRQMDRJTHS-FMDGEEDCSA-N
MAN
Query on MAN

Download Ideal Coordinates CCD File 
G [auth A]
H [auth A]
I [auth A]
J [auth A]
K [auth A]
G [auth A],
H [auth A],
I [auth A],
J [auth A],
K [auth A],
L [auth A],
M [auth A],
Q [auth B],
R [auth B],
S [auth B],
T [auth B],
U [auth B]
alpha-D-mannopyranose
C6 H12 O6
WQZGKKKJIJFFOK-PQMKYFCFSA-N
Biologically Interesting Molecules (External Reference) 1 Unique
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.30 Å
  • R-Value Free: 0.254 
  • R-Value Work: 0.204 
  • R-Value Observed: 0.206 
  • Space Group: P 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 49.54α = 77.33
b = 52.96β = 78.6
c = 66.54γ = 75.66
Software Package:
Software NamePurpose
REFMACrefinement
XDSdata reduction
SCALAdata scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-01-23
    Type: Initial release
  • Version 2.0: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Atomic model, Data collection, Derived calculations, Other, Structure summary
  • Version 2.1: 2023-12-20
    Changes: Data collection, Database references, Refinement description, Structure summary
  • Version 2.2: 2024-10-23
    Changes: Structure summary