4HST

Crystal structure of a double mutant of a class III engineered cephalosporin acylase

4HST の概要

• エントリーDOI: 10.2210/pdb4hst/pdb
• 関連するPDBエントリー: 4HSR
• 分子名称: glutaryl-7-aminocephalosporanic acid acylase alpha chain, glutaryl-7-aminocephalosporanic acid acylase beta chain, 5,5-dihydroxy-L-norvaline, ... (4 entities in total)
• 機能のキーワード: protein engineering, substrate specificity, transition state analogue, n-terminal hydrolase, hydrolase
• 由来する生物種: Pseudomonas; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 84231.23

構造登録者

Vrielink, A.,Golden, E.,Patterson, R.,Tie, W.J.,Anandan, A.,Flematti, G.,Molla, G.,Rosini, E.,Pollegioni, L. (登録日: 2012-10-30, 公開日: 2013-02-27, 最終更新日: 2024-02-28)

主引用文献

Golden, E.,Paterson, R.,Tie, W.J.,Anandan, A.,Flematti, G.,Molla, G.,Rosini, E.,Pollegioni, L.,Vrielink, A.
Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.
Biochem.J., 451:217-226, 2013

PubMed Abstract: The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1β), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

PubMed: 23373797
DOI: 10.1042/BJ20121715

実験手法

X-RAY DIFFRACTION (1.571 Å)