2J9Z

Tryptophan Synthase T110 mutant complex

Summary for 2J9Z

• Entry DOI: 10.2210/pdb2j9z/pdb
• Related: 1A50 1A5A 1A5B 1A5S 1BEU 1BKS 1C29 1C8V 1C9D 1CW2 1CX9 1FUY 1K3U 1K7E 1K7F 1K7X 1K8X 1K8Y 1K8Z 1KFB 1KFC 1KFE 1KFJ 1KFK 1QOP 1QOQ 1TJP 1TTP 1UBS 1WBJ 2CLE 2CLF 2CLI 2CLK 2CLL 2CLM 2CLO 2J9X 2J9Y 2TRS 2TSY 2TYS 2WSY
• Descriptor: TRYPTOPHAN SYNTHASE ALPHA CHAIN, TRYPTOPHAN SYNTHASE BETA CHAIN, PYRIDOXAL-5'-PHOSPHATE, ... (5 entities in total)
• Functional Keywords: aromatic amino acid biosynthesis, tryptophan biosynthesis, synthase carbon- oxygen lyase, amino-acid biosynthesis, lyase, allosteric enzyme, pyridoxal phosphate
• Biological source: SALMONELLA TYPHIMURIUM; More
• Total number of polymer chains: 2
• Total formula weight: 71871.81

Authors

Blumenstein, L.,Domratcheva, T.,Niks, D.,Ngo, H.,Seidel, R.,Dunn, M.F.,Schlichting, I. (deposition date: 2006-11-16, release date: 2007-12-04, Last modification date: 2011-07-13)

Primary citation

Blumenstein, L.,Domratcheva, T.,Niks, D.,Ngo, H.,Seidel, R.,Dunn, M.F.,Schlichting, I.
Betaq114N and Betat110V Mutations Reveal a Critically Important Role of the Substrate Alpha-Carboxylate Site in the Reaction Specificity of Tryptophan Synthase.
Biochemistry, 46:14100-, 2007

PubMed Abstract: 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.

PubMed: 18004874
DOI: 10.1021/BI7008568

Experimental method

X-RAY DIFFRACTION (1.8 Å)