微囊藻毒素降解酶MlrA的结构功能分析

doi:10.11949/0438-1157.20200729

摘要/Abstract

摘要：

MlrA（亦称microcystinase）是微囊藻毒素（microcystins， MCs）细菌降解途径中负责催化起始反应的关键蛋白酶，其结构特征与底物水解机制尚未明确。使用折叠识别法构建MlrA分子模型，通过分子对接和定点突变分析了酶-底物的结合方式与相互作用，结合蛋白重组表达对酶活性影响机制等进行了探究。结果表明MlrA是定位于细菌细胞质膜的整合膜蛋白，主要由8个跨膜α-螺旋（TM1~8）组成，功能结构域ABI（TM4~7）形成向周质空间开放的底物反应空腔。MlrA催化残基（E172、H205、H260和N264）位于膜内，其侧链投射至反应腔内部。微囊藻毒素LR （MC-LR）采用β-发夹构型与酶结合并将易裂键暴露于水分子附近，其水解机制为E172和H205通过一般碱催化将水分子去质子化激活，对Adda-Arg肽键羰基碳进行亲核攻击；接着H260和N264构成氧阴离子穴以稳定过渡态氧阴离子；最后H205或E172催化胺离去基团发生质子化，使四面体氧阴离子中间体崩解。此外，MlrA不是金属蛋白酶，无法与金属离子（Ⅱ）配位结合，菲咯啉类化合物使酶分子发生非特异性解折叠而失活，EDTA对底物结合位点具有竞争作用。本研究揭示了MlrA的属性与水解机制，为进一步探索MCs微生物降解机理提供一定参考依据。

关键词: 微囊藻毒素, 酶, 生物催化, 分子模拟, 酶促机制

Abstract:

The critical enzyme responsible for catalyzing the first step in the microcystin (MC) biodegradation pathway identified in bacterial strains is referred to as MlrA (also known as microcystinase). Its structural characteristics and substrate hydrolysis mechanism are not yet clear. In this paper, a molecular structure model of MlrA was established using a fold recognition method. Docking and site directed mutagenesis approach was employed to determine the binding modes and interaction network of enzyme-ligand complex. Factors affecting the catalytic activity of the enzyme were also elucidated by in vitro enzyme assays. The results show that MlrA is an integral membrane protein localized at the plasma membrane, its structure comprising mainly of eight transmembrane α-helices (TM1—8), in which the conserved ABI domain (TM4—7) forms a conical cavity with a large volume and opens to the periplasmic space. The catalytic residues (E172, H205, H260 and N264) are located into the membrane that projects their side chains into the cavity for catalysis. The MC-LR adopts a β-hairpin structure within a cavity to bind to MlrA, which then positions the scissile bond adjacent to a water molecule. Thus, a complete proteolytic mechanism of the enzyme was proposed. First, E172 and H205 general base-catalyzed deprotonation of a water molecule for nucleophilic attack on the Adda-Arg peptide bond. Then, H260 and N264, which form an oxyanion hole, stabilize the oxyanion transition state by hydrogen bonding. Finally, protonation of the amine leaving group of Adda-Arg peptide bond could be catalyzed by either H205 or E172 to allow collapse of the intermediate. Furthermore, this enzyme does not contain a metal-binding site (Ⅱ), its concentration-dependent inactivation by phenanthroline results from non-specific protein unfolding, EDTA competes for the binding site of the enzyme by developing an interaction network within the active site similar to that of the substrate. These findings suggest that MlrA is not a metalloproteinase. This study revealed the properties and hydrolysis mechanism of MlrA, and provided a reference for further exploration of the microbial degradation mechanism of MCs.

Key words: microcystins, enzyme, biocatalysis, molecular simulation, enzymatic mechanism

中图分类号:

潘禹, 王华生, 詹鸿峰, 孙缓缓, 范超, 刘祖文, 闫海. 微囊藻毒素降解酶MlrA的结构功能分析[J]. 化工学报, 2021, 72(3): 1643-1653.

PAN Yu, WANG Huasheng, ZHAN Hongfeng, SUN Huanhuan, FAN Chao, LIU Zuwen, YAN Hai. Structural and functional analysis of MlrA from microcystin-degrading bacteria[J]. CIESC Journal, 2021, 72(3): 1643-1653.

图/表 10

图1 MCs的结构通式

Fig.1 The general structure of microcystins

表1 MlrA点突变体的氨基酸残基及相应替代密码子

Table 1 Amino acid residues and respective codons exchanged in MlrA mutant studies

点突变体	DNA碱基变化
点突变体	原有密码子	替代密码子
E172A	GAA	GCA
E173A	GAA	GCA
R177A	CGC	GCC
W201A	TGG	GCG
H205A	CAT	GCT
H260A	CAC	GCC
H263A	CAT	GCT
N264A	AAC	GCC
E265A	GAA	GCA

图2 MlrA和MmRce1的二级结构比对

Fig.2 The alignment between MlrA and MmRce1 in secondary structure and residue levels

表2 不同Threading程序对齐结果模型的评估分析

Table 2 Evaluation analysis of the models form the alignment results from the different Threading programs

名称	最优模型编号^①	C_α-RSMD/?	对齐长度	TM-score^②
HHpred	93^#	1.95	227	0.86
pGenTHREADER	50^#	0.68	262	0.93
Phyre2	67^#	1.15	216	0.88
SPARKS-X	96^#	1.13	255	0.89
CEthreader	64^#	1.33	257	0.90

图3 MlrA分子模型质量检验结果

Fig.3 Validation results for the MlrA model

图4 MlrA分子模型的整体结构

Fig.4 The overall structure of the MlrA model

图5 MlrA与MC-LR的识别过程与催化机理(虚线表示氢键)

Fig.5 Mode of action and catalytic mechanism of MlrA for MC-LR(dashed bonds represent the hydrogen bond)

图6 MlrA与点突变体对MC-RR的催化活性

Fig.6 Biodegradation activity of MlrA and point mutants for MC-RR

图7 金属离子和螯合因子对MlrA活性的影响ND—检测到酶活力，但无法量化；NO—未检测到酶活性

Fig.7 Effects of metal ions and chelating factors on the activity of MlrA

图8 MlrA与EDTA的相互作用

Fig.8 The interaction networks between MlrA-EDTA complexes obtained by docking

参考文献 37

1	王小艺, 唐丽娜, 刘载文, 等. 城市湖库蓝藻水华形成机理[J]. 化工学报, 2012, 63(5): 1492-1497.
	Wang X Y, Tang L N, Liu Z W, et al. Formation mechanism of cyanobacteria bloom in urban lake reservoir[J]. CIESC Journal, 2012, 63(5): 1492-1497.
2	Paerl H W, Otten T G. Blooms bite the hand that feeds them[J]. Science, 2013, 342(6157): 433-434.
3	Terin U C, Sabogal-Paz L P. Microcystis aeruginosa and microcystin-LR removal by household slow sand filters operating in continuous and intermittent flows[J]. Water Research, 2019, 150: 29-39.
4	Svirčev Z, Drobac D, Tokodi N, et al. Toxicology of microcystins with reference to cases of human intoxications and epidemiological investigations of exposures to cyanobacteria and cyanotoxins[J]. Archives of Toxicology, 2017, 91(2): 621-650.
5	Zhu X Y, Shen Y T, Chen X G, et al. Biodegradation mechanism of microcystin-LR by a novel isolate of Rhizobium sp. TH and the evolutionary origin of the mlrA gene[J]. International Biodeterioration & Biodegradation, 2016, 115: 17-25.
6	Bourne D G, Riddles P, Jones G J, et al. Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR[J]. Environmental Toxicology, 2001, 16(6): 523-534.
7	Bourne D G, Jones G J, Blakeley R L, et al. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR[J]. Applied and Environmental Microbiology, 1996, 62(11): 4086-4094.
8	Dziga D, Lisznianska M, Wladyka B. Bioreactor study employing bacteria with enhanced activity toward cyanobacterial toxins microcystins[J]. Toxins, 2014, 6(8): 2379-2392.
9	Dexter J, Dziga D, Lv J, et al. Heterologous expression of mlrA in a photoautotrophic host-engineering cyanobacteria to degrade microcystins[J]. Environmental Pollution, 2018, 237: 926-935.
10	姜恬, 冯旭东, 李岩, 等. 底物特异性的生物催化与酶设计改造[J]. 化工进展, 2019, 38(1): 606-614.
	Jiang T, Feng X D, Li Y, et al. The biocatalysis and enzyme modification of substrate specificity[J]. Chemical Industry and Engineering Progress, 2019, 38(1): 606-614.
11	冯旭东, 吕波, 李春. 酶分子稳定性改造研究进展[J]. 化工学报, 2016, 67(1): 277-284.
	Feng X D, Lyu B, Li C. Advances in enzyme stability modification[J]. CIESC Journal, 2016, 67(1): 277-284.
12	孙建华, 戴荣继, 邓玉林. 酶固定化技术研究进展[J]. 化工进展, 2010, 29(4): 715-721.
	Sun J H, Dai R J, Deng Y L. Progress in enzyme immobilization technique[J]. Chemical Industry and Engineering Progress, 2010, 29(4): 715-721.
13	Söding J, Biegert A, Lupas A N. The HHpred interactive server for protein homology detection and structure prediction[J]. Nucleic Acids Research, 2005, 33(s2): W244-W248.
14	Lobley A, Sadowski M I, Jones D T. pGenTHREADER and pDomTHREADER: new methods for improved protein fold recognition and superfamily discrimination[J]. Bioinformatics, 2009, 25(14): 1761-1767.
15	Kelley L A, Mezulis S, Yates C M, et al. The Phyre2 web portal for protein modeling, prediction and analysis[J]. Nature Protocols, 2015, 10(6): 845-858.
16	Yang Y, Faraggi E, Zhao H, et al. Improving protein fold recognition and template-based modeling by employing probabilistic-based matching between predicted one-dimensional structural properties of query and corresponding native properties of templates[J]. Bioinformatics, 2011, 27(15): 2076-2082.
17	Zheng W, Wuyun Q, Li Y, et al. Detecting distant-homology protein structures by aligning deep neural-network based contact maps[J]. PLoS Computational Biology, 2019, 15(10): e1007411.
18	Melo F, Sali A. Fold assessment for comparative protein structure modeling[J]. Protein Science, 2007, 16(11): 2412-2426.
19	Zhang Y, Skolnick J. TM-align: a protein structure alignment algorithm based on the TM-score[J]. Nucleic Acids Research, 2005, 33(7): 2302-2309.
20	Laskowski R A, Macarthur M W, Moss D S, et al. PROCHECK: a program to check the stereochemical quality of protein structures[J]. Journal of Applied Crystallography, 1993, 26(2): 283-291.
21	Wiederstein M, Sippl M J. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins[J]. Nucleic Acids Research, 2007, 35(s2): W407-W410.
22	Colovos C, Yeates T O. Verification of protein structures: patterns of nonbonded atomic interactions[J]. Protein Science, 1993, 2(9): 1511-1519.
23	Morris G M, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility[J]. Journal of Computational Chemistry, 2009, 30(16): 2785-2791.
24	Yan H, Wang H S, Wang J F, et al. Cloning and expression of the first gene for biodegrading microcystin LR by Sphingopyxis sp. USTB-05[J]. Journal of Environmental Sciences, 2012, 24(10): 1816-1822.
25	Dziga D, Wladyka B, Zielińska G, et al. Heterologous expression and characterisation of microcystinase[J]. Toxicon, 2012, 59(5): 578-586.
26	Pei J, Mitchell D A, Dixon J E, et al. Expansion of type Ⅱ CAAX proteases reveals evolutionary origin of γ-secretase subunit APH-1[J]. Journal of Molecular Biology, 2011, 410(1): 18-26.
27	Manolaridis I, Kulkarni K, Dodd R B, et al. Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1[J]. Nature, 2013, 504(7479): 301-305.
28	Hampton S E, Dore T M, Schmidt W K. Rce1: mechanism and inhibition[J]. Critical Reviews in Biochemistry and Molecular Biology, 2018, 53(2): 157-174.
29	Khor B Y, Tye G J, Lim T S, et al. General overview on structure prediction of twilight-zone proteins[J]. Theoretical Biology and Medical Modelling, 2015, 12(1): 15-25.
30	Schoonman M J L, Knegtel R M A, Grootenhuis P D J. Practical evaluation of comparative modelling and threading methods[J]. Computers & Chemistry, 1998, 22(5): 369-375.
31	Geralt M, Alimenti C, Vallesi A, et al. Thermodynamic stability of psychrophilic and mesophilic pheromones of the protozoan ciliate Euplotes[J]. Biology, 2013, 2(1): 142-150.
32	Dalal V, Kumar P, Rakhaminov G, et al. Repurposing an ancient protein core structure: structural studies on FmtA, a novel esterase of Staphylococcus aureus[J]. Journal of Molecular Biology, 2019, 431(17): 3107-3123.
33	Buller A R, Townsend C A. Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad[J]. Proceedings of the National Academy of Sciences, 2013, 110(8): E653-E661.
34	Plummer L J, Hildebrandt E R, Porter S B, et al. Mutational analysis of the Ras converting enzyme reveals a requirement for glutamate and histidine residues[J]. Journal of Biological Chemistry, 2006, 281(8): 4596-4605.
35	Dolence J M, Steward L E, Dolence E K, et al. Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p[J]. Biochemistry, 2000, 39(14): 4096-4104.
36	Carvajal N, Orellana M S, Bórquez J, et al. Non-chelating inhibition of the H101N variant of human liver arginase by EDTA[J]. Journal of Inorganic Biochemistry, 2004, 98(8): 1465-1469.
37	Lopata A, Jójárt B, Surányi E V, et al. Beyond chelation: EDTA tightly binds Taq DNA polymerase, MutT and dUTPase and directly inhibits dNTPase activity[J]. Biomolecules, 2019, 9(10): 621-639.

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