Kim K K, Song H K, Shin D H, Hwang K Y, Suh S W
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
Structure. 1997 Feb 15;5(2):173-85. doi: 10.1016/s0969-2126(97)00177-9.
. Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms. True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface. Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions. Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals. Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity. This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources.
. We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively.
. PcL exhibits some structural features found in other lipases. The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases. The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface. The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site. This is similar to the mechanism described for other well characterized fungal and mammalian lipases.
脂肪酶是一类催化甘油三酯水解的酶，广泛分布于许多生物体中。真正的脂肪酶与酯酶的区别在于它们在油水界面表现出的特征性界面活化作用。脂肪酶是在温和条件下进行有机反应最常用的生物催化剂之一。它们的生物技术应用包括食品和油脂加工以及制备用于合成对映体纯药物的手性中间体。最近对几种脂肪酶的结构研究为理解水解活性、界面活化和立体选择性机制提供了一些线索。进行这项研究是为了提供有关细菌脂肪酶的结构信息，与其他来源的酶相比，这方面的信息相对有限。
我们利用X射线晶体学确定了洋葱伯克霍尔德菌（PcL）的三酰基甘油脂肪酶在无结合抑制剂情况下的晶体结构。该结构显示脂肪酶含有α/β水解酶折叠以及由Ser87、His286和Asp264残基组成的催化三联体。该酶与来自稻谷假单胞菌（PgL）和粘性色杆菌（CvL）的同源脂肪酶具有几个结构特征，包括一个钙结合位点。PcL的当前结构显示出一种高度开放的构象，活性位点可被溶剂接触。这与PgL和PcL的结构形成对比，在PgL和PcL中活性位点分别被封闭或部分打开的“盖子”掩埋。
PcL表现出其他脂肪酶中发现的一些结构特征。Ser-His-Asp催化三联体、氧阴离子孔的存在以及螺旋盖子的打开表明该酶与其他脂肪酶具有相同的催化和界面活化机制。在本研究中观察到的高度开放构象可能反映了脂肪酶在油水界面的活化形式。该结构表明细菌脂肪酶的界面活化涉及二级结构的重组以及盖子的大幅移动以暴露活性位点。这与其他特征明确的真菌和哺乳动物脂肪酶所描述的机制相似。
