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| - | [[Image:1rm4.gif|left|200px]] | + | {{Seed}} |
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| | {{STRUCTURE_1rm4| PDB=1rm4 | SCENE= }} | | {{STRUCTURE_1rm4| PDB=1rm4 | SCENE= }} |
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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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| - | ==Overview==
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| - | 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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_15236965}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15236965 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_15236965}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Gapdh-nadp complex]] | | [[Category: Gapdh-nadp complex]] |
| | [[Category: Rossmann fold]] | | [[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 Mon Jul 28 20:07:48 2008'' |
Revision as of 17:07, 28 July 2008
Template:STRUCTURE 1rm4
Crystal structure of recombinant photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP
Template:ABSTRACT PUBMED 15236965
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
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