Garba Lawal, Mohamad Yussoff Mohamad Ariff, Abd Halim Khairul Bariyyah, Ishak Siti Nor Hasmah, Mohamad Ali Mohd Shukuri, Oslan Siti Nurbaya, Raja Abd Rahman Raja Noor Zaliha
Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
PeerJ. 2018 Mar 19;6:e4347. doi: 10.7717/peerj.4347. eCollection 2018.
Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerant sp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed in . In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerant sp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with the activity of the Δ9-fatty acid desaturase on the membrane phospholipids.
膜结合脂肪酸去饱和酶进行氧化去饱和反应，以区域选择性和立体选择性方式在脂肪酰链内插入双键。Δ9-脂肪酸去饱和酶严格在大多数饱和底物的C9和10位之间形成第一个双键。由于细菌膜脂肪酸去饱和酶的三维结构尚不可得，有关这些酶的相关信息来自它们的氨基酸序列、定点诱变以及在类似膜结合去饱和酶中的结构域交换。耐寒的 sp. AMS8被发现能在低温下产生大量单不饱和脂肪酸。随后，一种活性Δ9-脂肪酸去饱和酶被分离并在 中功能性表达。据我们所知，本文首次报道了来自耐寒的 sp. AMS8的Δ9-脂肪酸去饱和酶的同源建模和对接研究。使用MODELLER 9.18版本并借助合适的模板构建了该酶的三维结构。该蛋白质模型包含所有膜结合去饱和酶催化活性所特有的三个保守组氨酸残基。对该结构进行了能量最小化，并使用拉氏图和ERRAT检查其正确性，结果分别显示该模型质量良好，得分分别为91.6%和65.0%。分别使用GROMACS 5版本中的CHARMM36力场和Autodock工具4.2版本，将该蛋白质模型用于进行MD模拟和棕榈酸的对接。结合能最低为 -6.8 kcal/mol的对接模拟中有许多残基与对接的棕榈酸紧密接触，即Ile26、Tyr95、Val179、Gly180、Pro64、Glu203、His34、His206、His71、Arg182、Thr85、Lys98和His177。有趣的是，在结合残基中分别有来自第一个、第二个和第三个保守组氨酸基序的His34、His71和His206，它们构成了该酶的活性位点。所得结果与Δ9-脂肪酸去饱和酶对膜磷脂的 活性相符。
