RCSB PDB - 3TW7: Structure of Rhizobium etli pyruvate carboxylase T882A crystallized without acetyl coenzyme-A

 3TW7

Structure of Rhizobium etli pyruvate carboxylase T882A crystallized without acetyl coenzyme-A


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.10 Å
  • R-Value Free: 0.292 
  • R-Value Work: 0.250 
  • R-Value Observed: 0.252 

wwPDB Validation   3D Report Full Report


This is version 1.1 of the entry. See complete history


Literature

Interaction between the biotin carboxyl carrier domain and the biotin carboxylase domain in pyruvate carboxylase from Rhizobium etli.

Lietzan, A.D.Menefee, A.L.Zeczycki, T.N.Kumar, S.Attwood, P.V.Wallace, J.C.Cleland, W.W.St Maurice, M.

(2011) Biochemistry 50: 9708-9723

  • DOI: https://doi.org/10.1021/bi201277j
  • Primary Citation of Related Structures:  
    3TW6, 3TW7

  • PubMed Abstract: 

    Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To effect catalysis, the tethered biotin of PC must gain access to active sites in both the biotin carboxylase domain and the carboxyl transferase domain. Previous studies have demonstrated that a mutation of threonine 882 to alanine in PC from Rhizobium etli renders the carboxyl transferase domain inactive and favors the positioning of biotin in the biotin carboxylase domain. We report the 2.4 Å resolution X-ray crystal structure of the Rhizobium etli PC T882A mutant which reveals the first high-resolution description of the domain interaction between the biotin carboxyl carrier protein domain and the biotin carboxylase domain. The overall quaternary arrangement of Rhizobium etli PC remains highly asymmetrical and is independent of the presence of allosteric activator. While biotin is observed in the biotin carboxylase domain, its access to the active site is precluded by the interaction between Arg353 and Glu248, revealing a mechanism for regulating carboxybiotin access to the BC domain active site. The binding location for the biotin carboxyl carrier protein domain demonstrates that tethered biotin cannot bind in the biotin carboxylase domain active site in the same orientation as free biotin, helping to explain the difference in catalysis observed between tethered biotin and free biotin substrates in biotin carboxylase enzymes. Electron density located in the biotin carboxylase domain active site is assigned to phosphonoacetate, offering a probable location for the putative carboxyphosphate intermediate formed during biotin carboxylation. The insights gained from the T882A Rhizobium etli PC crystal structure provide a new series of catalytic snapshots in PC and offer a revised perspective on catalysis in the biotin-dependent enzyme family.


  • Organizational Affiliation

    Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53201, United States.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Pyruvate carboxylase protein
A, B
1,165Rhizobium etli CFN 42Mutation(s): 1 
Gene Names: pycRHE_CH04002
EC: 6.4.1.1
UniProt
Find proteins for Q2K340 (Rhizobium etli (strain ATCC 51251 / DSM 11541 / JCM 21823 / NBRC 15573 / CFN 42))
Explore Q2K340 
Go to UniProtKB:  Q2K340
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ2K340
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
KCX
Query on KCX
A, B
L-PEPTIDE LINKINGC7 H14 N2 O4LYS
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.10 Å
  • R-Value Free: 0.292 
  • R-Value Work: 0.250 
  • R-Value Observed: 0.252 
  • Space Group: I 41
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 264.158α = 90
b = 264.158β = 90
c = 91.79γ = 90
Software Package:
Software NamePurpose
PHASERphasing
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2011-10-12
    Type: Initial release
  • Version 1.1: 2011-12-28
    Changes: Database references