5AP9

Controlled lid-opening in Thermomyces lanuginosus lipase - a switch for activity and binding

5AP9 の概要

• エントリーDOI: 10.2210/pdb5ap9/pdb
• 分子名称: LIPASE, 2-acetamido-2-deoxy-beta-D-glucopyranose, GLYCEROL, ... (6 entities in total)
• 機能のキーワード: hydrolase, thermomyces lanuginosus lipase, engineered disulfide bridge, controlled binding, dual switch, controlled activity
• 由来する生物種: THERMOMYCES LANUGINOSUS
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 59252.49

構造登録者

Skjold-Joergensen, J.,Vind, J.,Moroz, O.V.,Blagova, E.V.,Bhatia, V.K.,Svendsen, A.,Wilson, K.S.,Bjerrum, M.J. (登録日: 2015-09-15, 公開日: 2016-09-28, 最終更新日: 2024-11-13)

主引用文献

Skjold-Jrgensen, J.,Vind, J.,Moroz, O.V.,Blagova, E.,Bhatia, V.K.,Svendsen, A.,Wilson, K.S.,Bjerrum, M.J.
Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function.
Biochim. Biophys. Acta, 1865:20-27, 2017

PubMed Abstract: Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.

PubMed: 27693248
DOI: 10.1016/j.bbapap.2016.09.016

実験手法

X-RAY DIFFRACTION (1.8 Å)