1UKS

Crystal structure of F183L/F259L mutant cyclodextrin glucanotransferase complexed with a pseudo-maltotetraose derived from acarbose


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.215 
  • R-Value Work: 0.162 
  • R-Value Observed: 0.165 

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Ligand Structure Quality Assessment 


This is version 2.2 of the entry. See complete history


Literature

Effects of Essential Carbohydrate/Aromatic Stacking Interaction with Tyr100 and Phe259 on Substrate Binding of Cyclodextrin Glycosyltransferase from Alkalophilic Bacillus sp. 1011

Haga, K.Kanai, R.Sakamoto, O.Aoyagi, M.Harata, K.Yamane, K.

(2003) J Biochem 134: 881-891

  • DOI: https://doi.org/10.1093/jb/mvg215
  • Primary Citation of Related Structures:  
    1UKQ, 1UKS, 1UKT

  • PubMed Abstract: 

    The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.


  • Organizational Affiliation

    Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Cyclomaltodextrin glucanotransferase
A, B
686Bacillus sp. 1011Mutation(s): 2 
EC: 2.4.1.19
UniProt
Find proteins for P05618 (Bacillus sp. (strain 1011))
Explore P05618 
Go to UniProtKB:  P05618
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP05618
Sequence Annotations
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  • Reference Sequence
Oligosaccharides

Help

Entity ID: 2
MoleculeChains Length2D Diagram Glycosylation3D Interactions
4,6-dideoxy-alpha-D-xylo-hexopyranose-(1-4)-beta-D-galactopyranose
C, D
2N/A
Glycosylation Resources
GlyTouCan:  G94251DX
GlyCosmos:  G94251DX
Small Molecules
Ligands 3 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
GLC
Query on GLC

Download Ideal Coordinates CCD File 
E [auth A],
I [auth B]
alpha-D-glucopyranose
C6 H12 O6
WQZGKKKJIJFFOK-DVKNGEFBSA-N
ACI
Query on ACI

Download Ideal Coordinates CCD File 
F [auth A],
J [auth B]
6-AMINO-4-HYDROXYMETHYL-CYCLOHEX-4-ENE-1,2,3-TRIOL
C7 H13 N O4
XPHOBMULWMGEBA-VZFHVOOUSA-N
CA
Query on CA

Download Ideal Coordinates CCD File 
G [auth A],
H [auth A],
K [auth B],
L [auth B]
CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Free: 0.215 
  • R-Value Work: 0.162 
  • R-Value Observed: 0.165 
  • Space Group: P 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 64.85α = 85.19
b = 74.53β = 104.86
c = 78.94γ = 101.04
Software Package:
Software NamePurpose
MADNESSdata collection
FFFEARdata reduction
X-PLORmodel building
X-PLORrefinement
MADNESSdata reduction
FFFEARdata scaling
X-PLORphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-02-24
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Source and taxonomy, Version format compliance
  • Version 1.3: 2017-10-04
    Changes: Refinement description
  • Version 2.0: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Advisory, Atomic model, Data collection, Derived calculations, Structure summary
  • Version 2.1: 2021-11-10
    Changes: Database references, Structure summary
  • Version 2.2: 2023-10-25
    Changes: Data collection, Refinement description