2j9z
From Proteopedia
(New page: 200px<br /><applet load="2j9z" size="350" color="white" frame="true" align="right" spinBox="true" caption="2j9z, resolution 1.80Å" /> '''TRYPTOPHAN SYNTHASE ...) |
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==Overview== | ==Overview== | ||
| - | In the PLP-requiring alpha2beta2 tryptophan synthase complex, recognition | + | In the PLP-requiring alpha2beta2 tryptophan synthase complex, recognition of the substrate l-Ser at the beta-site includes a loop structure (residues beta110-115) extensively H-bonded to the substrate alpha-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: betaThr110 --> Val, and betaGln114 --> Asn. The betaT110V mutation greatly impairs both catalytic activity in the beta-reaction, and allosteric communication between the alpha- and beta-sites. The crystal structure of the betaT110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair beta-site catalysis and the communication of allosteric signals between the alpha- and beta-sites. Purified betaQ114N consists of two species of mutant protein, one with a reddish color (lambdamax = 506 nm). The reddish species is unable to react with l-Ser. The second betaQ114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with lambdamax approximately 320 nm, followed by a slower conversion over 24 h to the species with lambdamax = 506 nm. The 320 and 506 nm species originate from conversion of the alpha-aminoacrylate external aldimine to the internal aldimine and alpha-aminoacrylate, followed by the nucleophilic attack of alpha-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of beta114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase. |
==About this Structure== | ==About this Structure== | ||
| - | 2J9Z is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Salmonella_typhimurium Salmonella typhimurium] with <scene name='pdbligand=NA:'>NA</scene> and <scene name='pdbligand=PLP:'>PLP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Tryptophan_synthase Tryptophan synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.20 4.2.1.20] Known structural/functional Sites: <scene name='pdbsite=AC1:Plp Binding Site For Chain B'>AC1</scene> and <scene name='pdbsite=AC2:Na Binding Site For Chain B'>AC2</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2J9Z OCA]. | + | 2J9Z is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Salmonella_typhimurium Salmonella typhimurium] with <scene name='pdbligand=NA:'>NA</scene> and <scene name='pdbligand=PLP:'>PLP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Tryptophan_synthase Tryptophan synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.20 4.2.1.20] Known structural/functional Sites: <scene name='pdbsite=AC1:Plp+Binding+Site+For+Chain+B'>AC1</scene> and <scene name='pdbsite=AC2:Na+Binding+Site+For+Chain+B'>AC2</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2J9Z OCA]. |
==Reference== | ==Reference== | ||
| - | + | BetaQ114N and betaT110V mutations reveal a critically important role of the substrate alpha-carboxylate site in the reaction specificity of tryptophan synthase., Blumenstein L, Domratcheva T, Niks D, Ngo H, Seidel R, Dunn MF, Schlichting I, Biochemistry. 2007 Dec 11;46(49):14100-16. Epub 2007 Nov 16. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=18004874 18004874] | |
[[Category: Protein complex]] | [[Category: Protein complex]] | ||
[[Category: Salmonella typhimurium]] | [[Category: Salmonella typhimurium]] | ||
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[[Category: Blumenstein, L.]] | [[Category: Blumenstein, L.]] | ||
[[Category: Domratcheva, T.]] | [[Category: Domratcheva, T.]] | ||
| - | [[Category: Dunn, M | + | [[Category: Dunn, M F.]] |
[[Category: Ngo, H.]] | [[Category: Ngo, H.]] | ||
[[Category: Niks, D.]] | [[Category: Niks, D.]] | ||
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[[Category: tryptophan biosynthesis]] | [[Category: tryptophan biosynthesis]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:00:58 2008'' |
Revision as of 16:01, 21 February 2008
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TRYPTOPHAN SYNTHASE T110 MUTANT COMPLEX
Overview
In the PLP-requiring alpha2beta2 tryptophan synthase complex, recognition of the substrate l-Ser at the beta-site includes a loop structure (residues beta110-115) extensively H-bonded to the substrate alpha-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: betaThr110 --> Val, and betaGln114 --> Asn. The betaT110V mutation greatly impairs both catalytic activity in the beta-reaction, and allosteric communication between the alpha- and beta-sites. The crystal structure of the betaT110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair beta-site catalysis and the communication of allosteric signals between the alpha- and beta-sites. Purified betaQ114N consists of two species of mutant protein, one with a reddish color (lambdamax = 506 nm). The reddish species is unable to react with l-Ser. The second betaQ114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with lambdamax approximately 320 nm, followed by a slower conversion over 24 h to the species with lambdamax = 506 nm. The 320 and 506 nm species originate from conversion of the alpha-aminoacrylate external aldimine to the internal aldimine and alpha-aminoacrylate, followed by the nucleophilic attack of alpha-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of beta114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase.
About this Structure
2J9Z is a Protein complex structure of sequences from Salmonella typhimurium with and as ligands. Active as Tryptophan synthase, with EC number 4.2.1.20 Known structural/functional Sites: and . Full crystallographic information is available from OCA.
Reference
BetaQ114N and betaT110V mutations reveal a critically important role of the substrate alpha-carboxylate site in the reaction specificity of tryptophan synthase., Blumenstein L, Domratcheva T, Niks D, Ngo H, Seidel R, Dunn MF, Schlichting I, Biochemistry. 2007 Dec 11;46(49):14100-16. Epub 2007 Nov 16. PMID:18004874
Page seeded by OCA on Thu Feb 21 18:00:58 2008
Categories: Protein complex | Salmonella typhimurium | Tryptophan synthase | Blumenstein, L. | Domratcheva, T. | Dunn, M F. | Ngo, H. | Niks, D. | Schlichting, I. | Seidel, R. | NA | PLP | Allosteric enzyme | Amino-acid biosynthesis | Aromatic amino acid biosynthesis | Lyase | Pyridoxal phosphate | Synthase carbon- oxygen lyase | Tryptophan biosynthesis
