6WQY

GH5-4 broad specificity endoglucanase from Bacteroides salanitronis


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.05 Å
  • R-Value Free: 0.145 
  • R-Value Work: 0.139 
  • R-Value Observed: 0.139 

wwPDB Validation   3D Report Full Report


This is version 1.2 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
Cellulase385Phocaeicola salanitronis DSM 18170Mutation(s): 0 
Gene Names: Bacsa_0886
EC: 3.2.1.4
UniProt
Find proteins for F0R2T3 (Phocaeicola salanitronis (strain DSM 18170 / JCM 13657 / BL78))
Explore F0R2T3 
Go to UniProtKB:  F0R2T3
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupF0R2T3
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.05 Å
  • R-Value Free: 0.145 
  • R-Value Work: 0.139 
  • R-Value Observed: 0.139 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 40.48α = 90
b = 92.31β = 90
c = 95.15γ = 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: 2020-11-18
    Type: Initial release
  • Version 1.1: 2021-02-17
    Changes: Database references
  • Version 1.2: 2023-10-18
    Changes: Data collection, Database references, Refinement description