1rm4
From Proteopedia
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[[Image:1rm4.gif|left|200px]] | [[Image:1rm4.gif|left|200px]] | ||
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'''Crystal structure of recombinant photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP''' | '''Crystal structure of recombinant photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP''' | ||
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==Reference== | ==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:[http://www.ncbi.nlm.nih.gov/pubmed/15236965 15236965] | 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:[http://www.ncbi.nlm.nih.gov/pubmed/15236965 15236965] | ||
| - | [[Category: Glyceraldehyde-3-phosphate dehydrogenase (NADP(+)) (phosphorylating)]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Spinacia oleracea]] | [[Category: Spinacia oleracea]] | ||
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[[Category: Sparla, F.]] | [[Category: Sparla, F.]] | ||
[[Category: Trost, P.]] | [[Category: Trost, P.]] | ||
| - | [[Category: | + | [[Category: Gapdh-nadp complex]] |
| - | [[Category: | + | [[Category: Rossmann fold]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 07:39:43 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 04:39, 3 May 2008
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. 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 Sat May 3 07:39:43 2008
