植物体系中甲酯酶的底物专一性的理论研究

doi:10.7503/cjcu20150674

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (11): 2283.doi: 10.7503/cjcu20150674

植物体系中甲酯酶的底物专一性的理论研究

钱萍^1,^2,³(), 赵楠⁴, 陈峰⁴, 郭鸿^2,³()

1. 山东农业大学化学与材料科学学院, 泰安 271018
2. 田纳西大学-诺克斯维尔生物化学&细胞&分子生物学系, 诺克斯维尔, 田纳西州 37996, 美国
3. 橡树岭国家实验室, 田纳西大学/橡树岭国家实验室分子生物物理学中心, 橡树岭, 田纳西州 37830, 美国
4. 田纳西大学-诺克斯维尔植物科学系, 诺克斯维尔, 田纳西州 37996, 美国

收稿日期:2015-08-24 出版日期:2015-11-10 发布日期:2015-10-23
作者简介:联系人简介: 郭鸿, 男, 博士, 教授, 博士生导师, 主要从事生物体系模拟与计算研究. E-mail:hguo1@utk.edu;钱萍, 女, 博士, 副教授, 主要从事理论与计算化学研究. E-mail:qianp@sdau.edu.cn
基金资助:
美国国家自然科学基金(批准号: 0817940)、国家留学基金(批准号: 201408370020)、国家自然科学基金(批准号: 20903063)和山东农业大学博士后基金(批准号: 76335)资助

Understanding Substrate Specificity of Related Plant Methylesterases(MESs) from Computational Investigations

QIAN Ping^1,^2,^3,^*(), ZHAO Nan⁴, CHEN Feng⁴, GUO Hong^2,^3,^*()

1. Chemistry and Material Science Faculty, Shandong Agricultural University, Tai’an 271018, China
2. Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996, USA
3. UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
4. Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA

Received:2015-08-24 Online:2015-11-10 Published:2015-10-23
Contact: QIAN Ping,GUO Hong E-mail:qianp@sdau.edu.cn;hguo1@utk.edu

摘要/Abstract

摘要：

酶对天然底物的高度专一性是酶的特点之一. 然而关于酶是如何对底物具有高度专一性以及识别能力, 我们的理解仍然缺乏. 本文以植物体系中发现的一组甲酯酶(MESs)对一些底物[包括水杨酸甲酯(MeSA), 茉莉酮酸甲酯(MeJA)和吲哚-3-乙酸甲酯(MeIAA)]的催化反应为例, 报道了同源建模和理论计算对茉莉酮酸甲酯酶(AtMES10)和水杨酸结合蛋白2(SABP2)的研究结果. 基于简单的锁-钥匙理论(底物与酶结合时不发生基团的碰撞或严重排斥), 以底物对接到酶的活性部位(即底物中—COO的一部分占据可被催化丝氨酸亲核进攻的位置) 为原则, 可以在空间上为酶对底物的专一性提供解释. 模拟结果表明, SABP2可对MeSA有高活性, 对MeJA和MeIAA有低或无活性; AtMES10可对MeJA有高活性, 而对MeSA和MeIAA有低或无活性, 这与实验结果相一致. 因此, 相关酶的结构预测与计算机模拟对了解酶的底物专一性具有重要的意义.

关键词: 酶催化, 底物专一性, 计算机模拟, 蛋白质结构预测, 甲酯酶

Abstract:

One of enzyme’s hallmarks is the high specificity to their natural substrates. But our understanding is still lacking concerning how enzymes could achieve high specificity and substrate discrimination. This is the case for a group of related methylesterases(MESs) identified in plants that catalyze reactions of different substrates, including methyl salicylate(MeSA), methyl jasmonate(MeJA) and methyl indole-3-acetate(MeIAA). In this work, a homology model was built for AtMES10(a MeJA esterase),and this model along with the X-ray structure of SABP2(a MeSA esterase) was used to understand their substrate specificity. It is shown that the specificity may be explained based on the simple Lock-and-Key Model(that is, the active site being complementary in shape to the substrate) along with the requirement that the —COO moiety involved in the reaction occupies a position allowing the nucleophilic attack by the catalytic serine(that is, in a reactive configuration). The active site of SABP2 appears not to be complementary in shape to MeJA, and this may lead to a low activity on MeJA. For AtMES10, certain bulky residues in SABP2 are replaced by relatively small residues, allowing the substrate to bind to the active site and to be catalyzed by the enzyme. The results are consistent with the substrate specificity of these two enzymes observed experimentally. Explanations are also provided for the lack of the activities of AtMES10 and SABP2 on MeIAA and the lack of the activity of AtMES10 on MeSA.

Key words: Enzyme catalysis, Substrate specificity, Computer modeling, Protein structure prediction, Methyleste-rase

中图分类号:

O641

TrendMD:

钱萍, 赵楠, 陈峰, 郭鸿. 植物体系中甲酯酶的底物专一性的理论研究. 高等学校化学学报, 2015, 36(11): 2283.

QIAN Ping, ZHAO Nan, CHEN Feng, GUO Hong. Understanding Substrate Specificity of Related Plant Methylesterases(MESs) from Computational Investigations. Chem. J. Chinese Universities, 2015, 36(11): 2283.

图/表 9

Table 1 Substrate specificities of enzymes*

Name	MelAA	MeSA	MeJA
SABP2	-	+	-
AtMES10	-	-	+

Fig.1 Acylation reaction mechanism of AtMES10 with the catalytic triad involving Ser96, His253 and Asp225 RC:reactant complex; TI: tetrahedral intermediate; AE: acyl-enzyme complex. AtMES10 has esterase activity on methyl jasmonate(MeJA), but no activity was observed on methyl salicylate(MeSA) or methyl indole-3-acetate(MeIAA).

Fig.2 Sequence alignment of SABP2 and AtMES10

Fig.3 Four different conformations of MeJA downloaded from the ZINC database 1: zinc_4654657; 2: zinc_4975331; 3: zinc_4975327; 4: zinc_4975334. The coordinates for the 3D structures can be obtained from ZINC database. The relative stabilities of the conformations are given in Table 2 with 1 and 2 the most and the second most stable conformations, respectively, based on quantum mechanical calculations.

Table 2 Single point energies of four conformers of MeJA in vacuum gas and water solven with the help of polarizable continuum model(PCM) calculated at the level of B3LYP/6-31G(d)//HF/6-31G(d)

Structure code	B3LYP/6-31G(d)//HF/6-31G(d)		PCM
Structure code	SPE/a.u.	ΔE^*/(kJ·mol^-1)	SPE/a.u.	ΔE^*/(kJ·mol^-1)
1	-733.0910853	0	-733.1037684	0
2	-733.0894165	4.39	-733.1024123	3.55
3	-733.086736	11.41	-733.0997755	10.49
4	-733.0857963	13.88	-733.0999091	10.12

Fig.4 Comparison of the homology model of AtMES10(color: orange) on the X-ray structure of SABP2(PDB ID: 1XKL; color: cyan) (A) Superposition of the homology model of AtMES10 on the X-ray structure of SABP2. RMSD=0.062 nm based on the main-chain atoms. SA is shown in space filling; (B) superposition of the catalytic triads for the AtMES10 model and 1XKL. SA is also shown; (C) superposition of some other residues at the binding sites for the AtMES10 model and 1XKL(cyan); (D) X-ray structure of SABP2(1XKL) in space filling. Trp131 and Met149(pink) are located on the surface, SA(grey) can be seen through a channel from the surface to the binding site, see Methods; (E) AtMES10 model in space filling, Trp131 and Met149 are replaced by Ala146 and Val164, respectively.

Fig.5 Docking configuration of 1(the most stable conformer) of MeJA to the active site of SABP2 and AtMES10 model (A) SABP2: bad contacts occur between the MeJA chain and Trp131 or Met149; (B) AtMES10: there are few bad contacts in this case; (C) the active site structure of the AtMES10-MeJA complex after energy minimization. The substrate is well positioned relative to the catalytic triad for the catalysis.

Fig.6 Docking configuration of 2(the second most stable conformer) of MeJA to the active site of SABP2 and AtMES10 model (A) SABP2: the chain of MeJA is located at a different location without severe bad contacts with the residues from the enzyme; (B) AtMES10: without severe bad contacts.

Fig.7 Two docking configurations of indole-3-acetic acid(IAA) to the active site of the AtMES10 model with the carboxylic acid moiety located at the reactive position relative to Ser96

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植物体系中甲酯酶的底物专一性的理论研究

Understanding Substrate Specificity of Related Plant Methylesterases(MESs) from Computational Investigations

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