Maršavelski Aleksandra, Sabljić Igor, Sugimori Daisuke, Kojić-Prodić Biserka
Faculty of Science, Department of Chemistry, University of Zagreb, Zagreb, Croatia.
Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala SE-75651, Sweden; Ruđer Bošković Institute, Zagreb, Croatia.
Int J Biol Macromol. 2020 Apr 27;158:222-230. doi: 10.1016/j.ijbiomac.2020.04.198.
Two extracellular enzymes of the SGNH hydrolase superfamily reveal highly homologous 3D structures, but act on different substrates; one is a true phospholipase A1 from Streptomyces albidoflavus (SaPLA, EC: 3.1.1.32, PDB code: 4HYQ), whereas the promiscuous enzyme from Streptomyces rimosus (SrLip, EC: 3.1.1.3, PDB code: 5MAL) exhibits lipase, phospholipase, esterase, thioesterase, and Tweenase activities. To get insight into binding modes of phospholipid and triglyceride substrates in both enzymes and understand their chain-length preferences, we opted for computational approach based on in silico prepared enzyme-substrate complexes. Docking procedure and molecular dynamics simulations at microsecond time scale were applied. The modelled complexes of SaPLA and SrLip enzymes revealed substrate accommodation: a) the acyl-chain attached to sn-1 position fits into the hydrophobic pocket, b) the acyl-chain attached to sn-2 position fits in the hydrophobic cleft, whereas c) the sn-3 bound acyl chain of the triglyceride or polar head of the glycerophospholipid fits into the binding groove. Moreover, our results pinpointed subtle amino acid differences in the hydrophobic pockets of these two enzymes which accommodate the acyl chain attached to sn-1 position of glycerol to be responsible for the chain length preference. Slight differences in the binding grooves of SaPLA and SrLip, which accommodate the acyl chain attached to sn-3 position are responsible for exclusive phospholipase and both phospholipase/lipase activities of these two enzymes, respectively. The results of modelling correlate with the experimentally obtained kinetic parameters given in the literature and are important for protein engineering that aims to obtain a variant of enzyme, which would preferably act on the substrate of interest.
SGNH水解酶超家族的两种细胞外酶具有高度同源的三维结构，但作用于不同的底物；一种是来自白黄链霉菌的真正的磷脂酶A1（SaPLA，EC：3.1.1.32，PDB编号：4HYQ），而来自龟裂链霉菌的混杂酶（SrLip，EC：3.1.1.3，PDB编号：5MAL）表现出脂肪酶、磷脂酶、酯酶、硫酯酶和吐温酯酶活性。为了深入了解这两种酶中磷脂和甘油三酯底物的结合模式，并了解它们对链长的偏好，我们选择了基于计算机模拟制备的酶-底物复合物的计算方法。应用了对接程序和微秒时间尺度的分子动力学模拟。SaPLA和SrLip酶的模拟复合物显示出底物容纳情况：a）连接在sn-1位的酰基链适合进入疏水口袋，b）连接在sn-2位的酰基链适合进入疏水裂缝，而c）甘油三酯的sn-3位结合的酰基链或甘油磷脂的极性头部适合进入结合凹槽。此外，我们的结果指出了这两种酶疏水口袋中细微的氨基酸差异，这些差异容纳连接在甘油sn-1位的酰基链，从而导致了链长偏好。SaPLA和SrLip结合凹槽的细微差异，分别容纳连接在sn-3位的酰基链，这是这两种酶分别具有专一性磷脂酶活性和磷脂酶/脂肪酶活性的原因。模拟结果与文献中实验获得的动力学参数相关，对于旨在获得一种优选作用于目标底物的酶变体的蛋白质工程具有重要意义。
