1S08

Crystal Structure of the D147N Mutant of 7,8-Diaminopelargonic Acid Synthase


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.227 
  • R-Value Work: 0.202 
  • R-Value Observed: 0.203 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis.

Sandmark, J.Eliot, A.C.Famm, K.Schneider, G.Kirsch, J.F.

(2004) Biochemistry 43: 1213-1222

  • DOI: https://doi.org/10.1021/bi0358059
  • Primary Citation of Related Structures:  
    1S06, 1S07, 1S08, 1S09, 1S0A

  • PubMed Abstract: 

    The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutations in the active site were constructed independently. The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme. Crystallographic analyses of these mutants do not show significant changes in the structure of the active site. The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis. The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction. Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site. The D147N mutant is catalytically inactive in both half-reactions. The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role. Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases. This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose. Decarboxylated SAM [S-adenosyl-(5')-3-methylthiopropylamine] reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor.


  • Organizational Affiliation

    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Adenosylmethionine-8-amino-7-oxononanoate aminotransferase
A, B
429Escherichia coliMutation(s): 2 
Gene Names: BIOAB0774
EC: 2.6.1.62
UniProt
Find proteins for P12995 (Escherichia coli (strain K12))
Explore P12995 
Go to UniProtKB:  P12995
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP12995
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
LLP
Query on LLP
A, B
L-PEPTIDE LINKINGC14 H22 N3 O7 PLYS
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.227 
  • R-Value Work: 0.202 
  • R-Value Observed: 0.203 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 58.08α = 90
b = 56.527β = 96.32
c = 120.993γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
CCP4data scaling
AMoREphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-03-23
    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: 2018-03-07
    Changes: Advisory, Data collection
  • Version 1.4: 2021-10-27
    Changes: Advisory, Database references, Derived calculations