1RM5

Crystal structure of mutant S188A of photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP

Summary for 1RM5

• Entry DOI: 10.2210/pdb1rm5/pdb
• Related: 1JN0 1NBO 1RM3 1RM4
• Descriptor: Glyceraldehyde 3-phosphate dehydrogenase A, SULFATE ION, NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, ... (4 entities in total)
• Functional Keywords: rossmann fold, gapdh-nadp complex, oxidoreductase
• Biological source: Spinacia oleracea (spinach)
• Cellular location: Plastid, chloroplast : P19866
• Total number of polymer chains: 3
• Total formula weight: 111918.07

Authors

Sparla, F.,Fermani, S.,Falini, G.,Ripamonti, A.,Sabatino, P.,Pupillo, P.,Trost, P. (deposition date: 2003-11-27, release date: 2004-07-27, Last modification date: 2024-10-09)

Primary citation

Sparla, F.,Fermani, S.,Falini, G.,Zaffagnini, M.,Ripamonti, A.,Sabatino, P.,Pupillo, P.,Trost, P.
Coenzyme Site-directed Mutants of Photosynthetic A(4)-GAPDH Show Selectively Reduced NADPH-dependent Catalysis, Similar to Regulatory AB-GAPDH Inhibited by Oxidized Thioredoxin
J.Mol.Biol., 340:1025-1037, 2004

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.

PubMed: 15236965
DOI: 10.1016/j.jmb.2004.06.005

Experimental method

X-RAY DIFFRACTION (2.1 Å)