2PKP

Crystal structure of 3-isopropylmalate dehydratase (leuD)from Methhanocaldococcus Jannaschii DSM2661 (MJ1271)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.248 
  • R-Value Work: 0.201 
  • R-Value Observed: 0.201 

wwPDB Validation   3D Report Full Report


This is version 1.2 of the entry. See complete history


Literature

Substrate specificity determinants of the methanogen homoaconitase enzyme: structure and function of the small subunit

Jeyakanthan, J.Drevland, R.M.Gayathri, D.R.Velmurugan, D.Shinkai, A.Kuramitsu, S.Yokoyama, S.Graham, D.E.

(2010) Biochemistry 49: 2687-2696

  • DOI: https://doi.org/10.1021/bi901766z
  • Primary Citation of Related Structures:  
    2PKP

  • PubMed Abstract: 

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of alpha-hydroxy acids to beta-hydroxy acids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of alpha,beta-dicarboxylates with hydrophobic gamma-chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length gamma-carboxylate groups. These enzymes' stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins lead to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, the small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between alpha2 and alpha3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These structural and kinetic results will help to engineer new stereospecific hydro-lyase enzymes for chemoenzymatic syntheses.


  • Organizational Affiliation

    Life Science Group, National Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu Science Park, Hsinch 30076, Taiwan.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Homoaconitase small subunit170Methanocaldococcus jannaschii DSM 2661Mutation(s): 0 
Gene Names: aksE
EC: 4.2.1.114
UniProt
Find proteins for Q58667 (Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440))
Explore Q58667 
Go to UniProtKB:  Q58667
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ58667
Sequence Annotations
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  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.248 
  • R-Value Work: 0.201 
  • R-Value Observed: 0.201 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 90.992α = 90
b = 108.283β = 90
c = 45.449γ = 90
Software Package:
Software NamePurpose
CNSrefinement
HKL-2000data collection
HKL-2000data reduction
SCALEPACKdata scaling
AMoREphasing

Structure Validation

View Full Validation Report



Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 2008-04-22
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2023-10-25
    Changes: Data collection, Database references, Derived calculations, Refinement description