4FLS

Crystal structure of Amylosucrase inactive double mutant F290K-E328Q from Neisseria polysaccharea in complex with sucrose.

4FLS の概要

• エントリーDOI: 10.2210/pdb4fls/pdb
• 関連するPDBエントリー: 1G5A 4FLO 4FLQ 4FLR
• 関連するBIRD辞書のPRD_ID: PRD_900003
• 分子名称: Amylosucrase, beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose, CHLORIDE ION, ... (5 entities in total)
• 機能のキーワード: beta/alpha-barrel, glycoside hydrolase, amylose synthesis, sucrose isomerization, glucosyltransferase carbohydrate, transferase
• 由来する生物種: Neisseria polysaccharea
• 細胞内の位置: Secreted: Q9ZEU2
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 72357.24

構造登録者

Guerin, F.,Champion, E.,Moulis, C.,Barbe, S.,Tran, T.H.,Morel, S.,Descroix, K.,Monsan, P.,Mulard, L.A.,Remaud-Simeon, M.,Andre, I.,Mourey, L.,Tranier, S. (登録日: 2012-06-15, 公開日: 2012-10-31, 最終更新日: 2023-09-13)

主引用文献

Champion, E.,Guerin, F.,Moulis, C.,Barbe, S.,Tran, T.H.,Morel, S.,Descroix, K.,Monsan, P.,Mourey, L.,Mulard, L.A.,Tranier, S.,Remaud-Simeon, M.,Andre, I.
Applying pairwise combinations of amino Acid mutations for sorting out highly efficient glucosylation tools for chemo-enzymatic synthesis of bacterial oligosaccharides.
J.Am.Chem.Soc., 134:18677-18688, 2012

PubMed Abstract: Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea , a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semirational sublibraries accounting in total for 20,000 variants, we report here the isolation of three double mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement toward both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor as compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.

PubMed: 23072374
DOI: 10.1021/ja306845b

実験手法

X-RAY DIFFRACTION (2.3 Å)