Structural dynamics and mechanistic action guided engineering of lipolytic enzymes
Structural dynamics and mechanistic action guided engineering of lipolytic enzymes
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DOI：
10.1002/jcb.30410
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发表时间：
2023-04-23
影响因子：
4
通讯作者：
Kumar, Rajender
中科院分区：
文献类型：
--
作者：
Kumar, Rajender
脂肪酶由于其底物特异性的多样性，在许多工业应用中已被确立为重要的生物催化剂。各种脂肪酶的三维晶体结构的数据的可用性提供了一个机会，调节其结构和功能方面的设计和工程更好的版本的脂肪酶。为了研究微生物脂肪酶的结构组成，利用先进的生物信息学和分子动力学模拟方法对微生物脂肪酶进行了结构分析。在序列和功能上不同的嗜碱和嗜热脂肪酶的功能特性进行了研究，以了解其结构的区别特征。枯草芽孢杆菌嗜碱脂肪酶（利帕）在环区Ala 132-Met 137发生构象变化，随后活性位点残基His 156出现两种构象，一种朝向活性位点亲核残基Ser 77，另一种远离活性位点Ser 77。有趣的是，利帕的活性位点更多地暴露于溶剂，并且可以与开放构象的采用相关，所述开放构象可能在催化过程中延伸并将活性位点区域暴露于溶剂。此外，分子动力学模拟的嗜热脂肪酶从海洋链霉菌（MAS 1）揭示了N-和C-末端区域的二硫键的作用，并确定了金属离子结合位点，有利于酶的稳定性。新的嗜热嗜碱脂肪酶可以被设计成将MAS 1的稳定性特征整合到嗜碱利帕中。这些结构水平的内在特性可以用于脂肪酶工程，以根据工业过程的要求修改脂肪酶的活性和稳定性。
Lipases have been established as important biocatalysts in several industrial applications, owing to their diverse substrate specificity. The availability of data on three-dimensional crystal structures for various lipases offers an opportunity for modulating their structural and functional aspects to design and engineer better versions of lipases. With the aim of investigating the structural components governing the extremophilic behavior of lipases, structural analysis of microbial lipases was performed using advanced bioinformatics and molecular dynamics simulation approaches. In sequences and functionally distinct alkaliphilic and thermophilic lipases were investigated for their functional properties to understand the distinguishing features of their structures. The alkaliphilic lipase from Bacillus subtilis (LipA) showed conformational changes in the loop region Ala132-Met137, subsequently, the active site residue His156 shows two conformations, toward the active site nucleophilic residues Ser77 and away from the Ser77. Interestingly, the active site of LipA is more solvent-exposed and can be correlated with the adoption of an open conformation which might extend and expose the active site region to solvents during the catalysis process. Furthermore, the MD simulation of thermophilic lipase from marine Streptomyces (MAS1) revealed the role of N- and C-terminal regions with disulfide bridges and identified a metal ion binding site that facilitates the enzyme stability. The novel thermo-alkaliphilic lipase can be designed to integrate the stability features of MAS1 into the alkaliphilic LipA. These structural-level intrinsic characteristics can be used for lipase engineering to amend the lipase activity and stability as per the requirements of the industrial processes.