5TPQ

E. coli alkaline phosphatase D101A, D153A, R166S, E322A, K328A mutant

5TPQ の概要

• エントリーDOI: 10.2210/pdb5tpq/pdb
• 関連するPDBエントリー: 3TG0
• 分子名称: Alkaline phosphatase, ZINC ION, PHOSPHATE ION, ... (4 entities in total)
• 機能のキーワード: alkaline phosphatase, generalist enzyme, bimetallo motif, catalytic promiscuity, evolution, d101a/d153a/r166s/e322a/k328a, hydrolase
• 由来する生物種: Escherichia coli (strain K12)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 93769.29

構造登録者

Sunden, F.,AlSadhan, I.,Lyubimov, A.Y.,Doukov, T.,Swan, J.,Herschlag, D. (登録日: 2016-10-20, 公開日: 2017-11-01, 最終更新日: 2024-10-30)

主引用文献

Sunden, F.,AlSadhan, I.,Lyubimov, A.,Doukov, T.,Swan, J.,Herschlag, D.
Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.
J. Biol. Chem., 292:20960-20974, 2017

PubMed Abstract: Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. We mutated distinguishing active-site residues to generate enzymes that had a common Zn bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ∼10-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10-10-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.

PubMed: 29070681
DOI: 10.1074/jbc.M117.788240

実験手法

X-RAY DIFFRACTION (2.45 Å)