1Q6D

Crystal structure of Soybean Beta-Amylase Mutant (M51T) with Increased pH Optimum


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.215 
  • R-Value Work: 0.177 
  • R-Value Observed: 0.177 

wwPDB Validation   3D Report Full Report


This is version 2.1 of the entry. See complete history


Literature

Structural and Enzymatic Analysis of Soybean {beta}-Amylase Mutants with Increased pH Optimum

Hirata, A.Adachi, M.Sekine, A.Kang, Y.N.Utsumi, S.Mikami, B.

(2004) J Biol Chem 279: 7287-7295

  • DOI: https://doi.org/10.1074/jbc.M309411200
  • Primary Citation of Related Structures:  
    1Q6C, 1Q6D, 1Q6E, 1Q6F, 1Q6G

  • PubMed Abstract: 

    Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.


  • Organizational Affiliation

    Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
beta-amylase495Glycine maxMutation(s): 1 
EC: 3.2.1.2
UniProt
Find proteins for P10538 (Glycine max)
Explore P10538 
Go to UniProtKB:  P10538
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP10538
Sequence Annotations
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  • Reference Sequence
Oligosaccharides

Help

Entity ID: 2
MoleculeChains Length2D Diagram Glycosylation3D Interactions
alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose
B
2N/A
Glycosylation Resources
GlyTouCan:  G07411ON
GlyCosmos:  G07411ON
Entity ID: 3
MoleculeChains Length2D Diagram Glycosylation3D Interactions
alpha-D-glucopyranose-(1-4)-beta-D-glucopyranose
C
2N/A
Glycosylation Resources
GlyTouCan:  G66120GK
GlyCosmos:  G66120GK
Biologically Interesting Molecules (External Reference) 2 Unique
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.215 
  • R-Value Work: 0.177 
  • R-Value Observed: 0.177 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 86.354α = 90
b = 86.354β = 90
c = 145.629γ = 120
Software Package:
Software NamePurpose
CNSrefinement
SAINTdata reduction
SAINTdata scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-02-24
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 2.0: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Atomic model, Data collection, Derived calculations, Structure summary
  • Version 2.1: 2021-11-10
    Changes: Database references, Structure summary