6XRK

GH5-4 broad specificity endoglucanase from an uncultured bovine rumen ciliate


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.42 Å
  • R-Value Free: 0.167 
  • R-Value Work: 0.145 
  • R-Value Observed: 0.145 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis.

Glasgow, E.M.Kemna, E.I.Bingman, C.A.Ing, N.Deng, K.Bianchetti, C.M.Takasuka, T.E.Northen, T.R.Fox, B.G.

(2020) J Biol Chem 295: 17752-17769

  • DOI: https://doi.org/10.1074/jbc.RA120.015328
  • Primary Citation of Related Structures:  
    4IM4, 6MQ4, 6PZ7, 6Q1I, 6UI3, 6WQP, 6WQV, 6WQY, 6XRK, 6XSO, 6XSU

  • PubMed Abstract: 

    Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.


  • Organizational Affiliation

    Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA; Great Lakes Bioenergy Research Center, Madison, Wisconsin, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Glycoside hydrolase type 1
A, B
374uncultured bovine rumen ciliateMutation(s): 0 
EC: 3
UniProt
Find proteins for G5DDB8 (uncultured bovine rumen ciliate)
Explore G5DDB8 
Go to UniProtKB:  G5DDB8
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupG5DDB8
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
BTB
Query on BTB

Download Ideal Coordinates CCD File 
D [auth A],
I [auth B]
2-[BIS-(2-HYDROXY-ETHYL)-AMINO]-2-HYDROXYMETHYL-PROPANE-1,3-DIOL
C8 H19 N O5
OWMVSZAMULFTJU-UHFFFAOYSA-N
PEG
Query on PEG

Download Ideal Coordinates CCD File 
J [auth B]DI(HYDROXYETHYL)ETHER
C4 H10 O3
MTHSVFCYNBDYFN-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
E [auth A]CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
MG
Query on MG

Download Ideal Coordinates CCD File 
C [auth A],
F [auth B],
G [auth B],
H [auth B]
MAGNESIUM ION
Mg
JLVVSXFLKOJNIY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.42 Å
  • R-Value Free: 0.167 
  • R-Value Work: 0.145 
  • R-Value Observed: 0.145 
  • Space Group: P 2 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 72.32α = 90
b = 84.99β = 90
c = 121.32γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Department of Energy (DOE, United States)United StatesDEFC0207ER64494
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesNIH 5 T32 GM008349 Biotechnology Training Program

Revision History  (Full details and data files)

  • Version 1.0: 2020-10-28
    Type: Initial release
  • Version 1.1: 2020-11-04
    Changes: Database references
  • Version 1.2: 2021-02-17
    Changes: Database references
  • Version 1.3: 2023-10-18
    Changes: Data collection, Database references, Refinement description