1M8J

Crystal Structure Of Methanobacterium Thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase Mutant R136A complexed with NAD


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.265 
  • R-Value Work: 0.225 
  • R-Value Observed: 0.225 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Mutational, Structural, and Kinetic Studies of the ATP-binding Site of Methanobacterium thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase

Saridakis, V.Pai, E.F.

(2003) J Biol Chem 278: 34356-34363

  • DOI: https://doi.org/10.1074/jbc.M205369200
  • Primary Citation of Related Structures:  
    1M8F, 1M8G, 1M8J, 1M8K

  • PubMed Abstract: 

    Several residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent Km values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. All four Arg mutants displayed unaltered Km values for NMN. The apparent kcat values of the R11K and R136K mutants were the same as those of WT NMNATase but the apparent kcat values of the alanine mutants had decreased. Crystal structures of the Arg mutants revealed NAD+ and SO42- molecules trapped at their active sites. The binding interactions of NAD+ were unchanged but the binding of SO42- was altered in these mutants compared with wild type. The alanine mutants at positions His-16 and His-19 retained approximately 6 and 1.3%, respectively, of WT NMNATase activity indicating that His-19 is a key catalytic group. Surprisingly, this H19A mutant displayed a novel and distinct mode of NAD+ binding when co-crystallized in the presence of NAD+ and SO42-.


  • Organizational Affiliation

    Molecular and Structural Biology, Ontario Cancer Institute, University Health Network, and the Department of Medical Biophysics, University of Toronto, Ontario, Canada.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Nicotinamide-nucleotide Adenylyltransferase181Methanothermobacter thermautotrophicusMutation(s): 1 
Gene Names: mth0150
EC: 2.7.7.1
UniProt
Find proteins for O26253 (Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H))
Explore O26253 
Go to UniProtKB:  O26253
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupO26253
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NAD
Query on NAD

Download Ideal Coordinates CCD File 
C [auth A]NICOTINAMIDE-ADENINE-DINUCLEOTIDE
C21 H27 N7 O14 P2
BAWFJGJZGIEFAR-NNYOXOHSSA-N
SO4
Query on SO4

Download Ideal Coordinates CCD File 
B [auth A]SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.265 
  • R-Value Work: 0.225 
  • R-Value Observed: 0.225 
  • Space Group: P 63 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 91.418α = 90
b = 91.418β = 90
c = 107.755γ = 120
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2003-07-15
    Type: Initial release
  • Version 1.1: 2008-04-28
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.4: 2024-02-14
    Changes: Data collection, Refinement description