1rm3: Difference between revisions
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[[Image:1rm3.gif|left|200px]] | [[Image:1rm3.gif|left|200px]] | ||
'''Crystal structure of mutant T33A of photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP''' | {{Structure | ||
|PDB= 1rm3 |SIZE=350|CAPTION= <scene name='initialview01'>1rm3</scene>, resolution 2.20Å | |||
|SITE= | |||
|LIGAND= <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene> and <scene name='pdbligand=NDP:NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE'>NDP</scene> | |||
|ACTIVITY= [http://en.wikipedia.org/wiki/Glyceraldehyde-3-phosphate_dehydrogenase_(NADP(+))_(phosphorylating) Glyceraldehyde-3-phosphate dehydrogenase (NADP(+)) (phosphorylating)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.2.1.13 1.2.1.13] | |||
|GENE= GapA ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=3562 Spinacia oleracea]) | |||
}} | |||
'''Crystal structure of mutant T33A of photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP''' | |||
==Overview== | ==Overview== | ||
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==About this Structure== | ==About this Structure== | ||
1RM3 is a [ | 1RM3 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Spinacia_oleracea Spinacia oleracea]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1RM3 OCA]. | ||
==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:// | 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: Glyceraldehyde-3-phosphate dehydrogenase (NADP(+)) (phosphorylating)]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
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[[Category: rossmann fold]] | [[Category: rossmann fold]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 13:53:26 2008'' | ||
Revision as of 14:53, 20 March 2008
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| , resolution 2.20Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | and | ||||||
| Gene: | GapA (Spinacia oleracea) | ||||||
| Activity: | Glyceraldehyde-3-phosphate dehydrogenase (NADP(+)) (phosphorylating), with EC number 1.2.1.13 | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
Crystal structure of mutant T33A of photosynthetic glyceraldehyde-3-phosphate dehydrogenase A4 isoform, complexed with NADP
OverviewOverview
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 StructureAbout this Structure
1RM3 is a Single protein structure of sequence from Spinacia oleracea. Full crystallographic information is available from OCA.
ReferenceReference
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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