1RM3

Crystal structure of mutant T33A of photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.237 
  • R-Value Work: 0.212 
  • R-Value Observed: 0.212 

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Ligand Structure Quality Assessment 


This is version 1.4 of the entry. See complete history


Literature

Coenzyme Site-directed Mutants of Photosynthetic A(4)-GAPDH Show Selectively Reduced NADPH-dependent Catalysis, Similar to Regulatory AB-GAPDH Inhibited by Oxidized Thioredoxin

Sparla, F.Fermani, S.Falini, G.Zaffagnini, M.Ripamonti, A.Sabatino, P.Pupillo, P.Trost, P.

(2004) J Mol Biol 340: 1025-1037

  • DOI: https://doi.org/10.1016/j.jmb.2004.06.005
  • Primary Citation of Related Structures:  
    1RM3, 1RM4, 1RM5

  • PubMed Abstract: 

    Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.


  • Organizational Affiliation

    Laboratorio di Fisiologia molecolare delle piante, Dipartimento di Biologia Evoluzionistica Sperimentale, via Irnerio 42, Università di Bologna, I-40126 Bologna, Italy.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Glyceraldehyde 3-phosphate dehydrogenase AA [auth O],
B [auth A],
C [auth B]
337Spinacia oleraceaMutation(s): 1 
Gene Names: GapA
EC: 1.2.1.13
UniProt
Find proteins for P19866 (Spinacia oleracea)
Explore P19866 
Go to UniProtKB:  P19866
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP19866
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NDP
Query on NDP

Download Ideal Coordinates CCD File 
H [auth O],
M [auth A],
Q [auth B]
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
C21 H30 N7 O17 P3
ACFIXJIJDZMPPO-NNYOXOHSSA-N
SO4
Query on SO4

Download Ideal Coordinates CCD File 
D [auth O]
E [auth O]
F [auth O]
G [auth O]
I [auth A]
D [auth O],
E [auth O],
F [auth O],
G [auth O],
I [auth A],
J [auth A],
K [auth A],
L [auth A],
N [auth B],
O [auth B],
P [auth B]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.237 
  • R-Value Work: 0.212 
  • R-Value Observed: 0.212 
  • Space Group: C 2 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 142.246α = 90
b = 185.045β = 90
c = 106.282γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
CNSrefinement

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-07-27
    Type: Initial release
  • Version 1.1: 2008-04-29
    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: 2023-08-23
    Changes: Data collection, Refinement description