1NS8

Crystal structure of galactose mutarotase from Lactococcus lactis mutant D243N complexed with galactose


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.175 
  • R-Value Observed: 0.178 

Starting Model: experimental
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This is version 1.6 of the entry. See complete history


Literature

The Catalytic Mechanism of Galactose Mutarotase

Thoden, J.B.Kim, J.Raushel, F.M.Holden, H.M.

(2003) Protein Sci 12: 1051-1059

  • DOI: https://doi.org/10.1110/ps.0243203
  • Primary Citation of Related Structures:  
    1NS0, 1NS2, 1NS4, 1NS7, 1NS8, 1NSM, 1NSR, 1NSS, 1NSU, 1NSV, 1NSX, 1NSZ

  • PubMed Abstract: 

    Galactose mutarotase catalyzes the first step in normal galactose metabolism by catalyzing the conversion of beta-D-galactose to alpha-D-galactose. The structure of the enzyme from Lactococcus lactis was recently solved in this laboratory and shown to be topologically similar to domain 5 of beta-galactosidase. From this initial X-ray analysis, four amino acid residues were demonstrated to be intimately involved in sugar binding to the protein: His 96, His 170, Asp 243, and Glu 304. Here we present a combined X-ray crystallographic and kinetic analysis designed to examine the role of these residues in the reaction mechanism of the enzyme. For this investigation, the following site-directed mutant proteins were prepared: H96N, H170N, D243N, D243A, E304Q, and E304A. All of the structures of these proteins, complexed with either glucose or galactose, were solved to a nominal resolution of 1.95 A or better, and their kinetic parameters were measured against D-galactose, D-glucose, L-arabinose, or D-xylose. From these studies, it can be concluded that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243 are important for proper substrate positioning within the active site. Specifically, Glu 304 serves as the active site base to initiate the reaction by removing the proton from the C-1 hydroxyl group of the sugar substrate and His 170 functions as the active site acid to protonate the C-5 ring oxygen.


  • Organizational Affiliation

    Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544, USA. [email protected]


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
GALACTOSE MUTAROTASE
A, B
347Lactococcus lactisMutation(s): 1 
EC: 5.1.3.3
UniProt
Find proteins for Q9ZB17 (Lactococcus lactis)
Explore Q9ZB17 
Go to UniProtKB:  Q9ZB17
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9ZB17
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.175 
  • R-Value Observed: 0.178 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 44.8α = 90
b = 76.4β = 90
c = 211.1γ = 90
Software Package:
Software NamePurpose
FRAMBOdata collection
SAINTdata reduction
TNTrefinement
SAINTdata scaling
TNTphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-02-11
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-10-11
    Changes: Refinement description
  • Version 1.4: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Data collection, Database references, Derived calculations, Structure summary
  • Version 1.5: 2021-10-27
    Changes: Database references, Structure summary
  • Version 1.6: 2023-08-16
    Changes: Data collection, Refinement description