Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark; Novozymes A/S, Brudelysvej 35, DK-2880 Bagværd, Denmark.
Novozymes A/S, Brudelysvej 35, DK-2880 Bagværd, Denmark.
Biochim Biophys Acta Proteins Proteom. 2017 Jan;1865(1):20-27. doi: 10.1016/j.bbapap.2016.09.016. Epub 2016 Sep 28.
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.
在这里，我们展示了一种含有生化开关的脂肪酶突变体，该开关允许盖子的打开和关闭独立于环境进行控制。与激活控制开关诱导盖子打开后观察到的结合相比，TlL 突变体的封闭形式显示与疏水性表面的低结合。我们直接表明，这种突变体的脂质结合与开放的盖子构象有关，证明了暴露的氨基酸残基的影响及其在水 - 脂质界面的结合参与。开关是通过在 86 和 255 位将两个半胱氨酸残基引入蛋白质骨架而创建的。突变体的晶体结构显示 C86 和 C255 之间成功形成了二硫键，这导致盖子结构域的张力闭合。在底物乳液和天然脂质层上证明了对酶活性和结合的控制。与野生型相比，锁定形式的酶活性（约 10%）较低。释放锁定后，酶活性得到完全恢复。与野生型相比，锁定脂肪酶对天然脂质底物的结合仅为 10%，但添加还原剂后结合恢复。QCM-D 测量显示未锁定脂肪酶的结合速率增加了七倍。TlL_locked 突变体显示整个蛋白质的结构变化，这对于理解盖子打开和关闭的机制很重要。我们的实验结果揭示了感兴趣的位点，可用于未来的诱变研究，旨在改变 TlL 的激活机制，并为生成在受控条件下激活的可调谐脂肪酶创造了前景。
