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1rm4
From Proteopedia
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Crystal structure of recombinant photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP
Overview
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.
About this Structure
1RM4 is a Single protein structure of sequence from Spinacia oleracea with SO4 and NDP as ligands. Active as Glyceraldehyde-3-phosphate dehydrogenase (NADP(+)) (phosphorylating), with EC number 1.2.1.13 Full crystallographic information is available from OCA.
Reference
Coenzyme site-directed mutants of photosynthetic A4-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, J Mol Biol. 2004 Jul 23;340(5):1025-37. PMID:15236965
Page seeded by OCA on Wed Nov 21 01:42:20 2007
