微囊藻毒素降解酶MlrA的生物学特征及催化机理研究进展

doi:10.11949/0438-1157.20191008

摘要/Abstract

摘要：

蓝藻水华产生并释放的微囊藻毒素（microcystins, MCs）在自然环境中高度稳定，难以被传统水处理技术有效去除，对饮用水安全及生态系统构成严重威胁。通过微生物代谢作用，可实现MCs的高效原位酶促降解。目前，已从天然水体及其沉积物中表征部分具有高效降解MCs能力的细菌，且在酶促途径中鉴别出负责催化起始反应的关键水解酶MlrA（即microcystinase）。本文在阐明MlrA进化关系的基础上，就该酶的活性特征、分子结构与催化机理进行了综述，并对MlrA的研究方向进行了展望，以期为水环境的生物修复提供参考。

关键词: 微囊藻毒素, 酶, 生物催化, MlrA, 分子生物学

Abstract:

The microcystins (MCs) produced and released by cyanobacteria blooms are highly stable in the natural environment and are difficult to be effectively removed by traditional water treatment technologies, posing a serious threat to drinking water safety and ecosystems. Microbial biodegradation is an effective strategy where microbial metabolic potential can be harnessed forin-situ andex-situ detoxification of MCs. At present, many studies have shown that some bacteria isolated from water bodies and sediments are capable of degrading MCs, which as an efficient and environmentally friendly strategy for MCs removal. The critical enzyme identified in bacterial strains is referred to as MlrA (also known as microcystinase), play a major role in the water body bioremediation process, responsible for catalyzing the first step reaction in the MCs biodegradation pathway. In this paper, based on evolutionary relationships of MlrA, reviews the characteristics of enzyme activity, molecular structure and catalytic mechanism of this enzyme. The further research subjects of MlrA were also proposed. This review could provide a reference for bioremediation of contaminated water environment.

Key words: microcystins, enzyme, biocatalysis, microcystinase, molecular biology

中图分类号:

Q 71

潘禹, 王华生, 詹鸿峰, 孙缓缓. 微囊藻毒素降解酶MlrA的生物学特征及催化机理研究进展[J]. 化工学报, 2020, 71(3): 945-954.

Yu PAN, Huasheng WANG, Hongfeng ZHAN, Huanhuan SUN. Progress of biological characterization and mechanism of enzymatic degradation of microcystinase[J]. CIESC Journal, 2020, 71(3): 945-954.

图/表 5

图1 MCs的化学结构式

Fig.1 General chemical structure of MCs

图2 不同菌株来源MlrA酶的氨基酸序列比对

Fig.2 Sequence alignment of MlrA from different strains

图3 MmRce1（a）与MlrA（b）的分子结构

Fig.3 Three dimensional structure ofMmRce1 (a) and MlrA (b)

图4 MlrA的催化位点（深色虚线标记关键残基之间的N-O距离；H260和N264之间的氢键显示为浅色虚线）

Fig.4 Crystal structure of MlrA catalytic site

图5 MlrA的结构域组成

Fig.5 Domain organisation of MlrA

参考文献 68

1	Rastogi R P,Sinha R P,Incharoensakdi A.The cyanotoxin-microcystins: current overview[J].Reviews in Environmental Science and Bio/Technology,2014,13(2):215-249.
2	Yaw M I,Fei Y,Zhen D,et al.Exposure routes and health effects of microcystins on animals and humans: a mini-review[J].Toxicon,2018,151:156-162.
3	Fontanillo M,Köhn M.Microcystins: synthesis and structure-activity relationship studies toward PP1 and PP2A[J].Bioorganic and Medicinal Chemistry,2018,26(6):1118-1126.
4	Douglas P,Moorhead G B G,Ye R,et al.Protein phosphatases regulate DNA-dependent protein kinase activity[J].Journal of Biological Chemistry,2001,276(22):18992-18998.
5	Ding W X,Ong C N.Role of oxidative stress and mitochondrial changes in cyanobacteria-induced apoptosis and hepatotoxicity[J].FEMS Microbiology Letters,2003,220(1):1-7.
6	La-Salete R,Oliveira M M,Palmeira C A,et al.Mitochondria a key role in microcystin-LR kidney intoxication[J].Journal of Applied Toxicology,2008,28(1):55-62.
7	Ho L,Sawade E,Newcombe G.Biological treatment options for cyanobacteria metabolite removal—a review[J].Water Research,2012,46(5):1536-1548.
8	闫海,张超,魏巍,等.微囊藻毒素生物降解的研究进展[J].环境工程学报,2007,1(10):8-12.
	Yan H,Zhang C,Wei W,et al.Advances in the biodegradation of microcystins[J].Chinese Journal of Environmental Engineering,2007,1(10):8-12.
9	Li J M,Li R H,Li J.Current research scenario for microcystins biodegradation — a review on fundamental knowledge, application prospects and challenges[J].Science of the Total Environment,2017,595:615-632.
10	Dziga D,Wasylewski M,Wladyka B,et al.Microbial degradation of microcystins[J].Chemical Research in Toxicology,2013,26(6):841-852.
11	Harada K,Imanishi S,Kato H,et al.Isolation of Adda from microcystin-LR by microbial degradation[J].Toxicon,2004,44(1):107-109.
12	Jones G J,Bourne D G,Blakeley R L,et al.Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria[J].Natural Toxins,1994,2(4):228-235.
13	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.
14	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.
15	Zhu X,Shen Y,Chen X,et al.Biodegradation mechanism of microcystin-LR by a novel isolate ofRhizobium sp. TH and the evolutionary origin of themlrA gene[J].International Biodeterioration and Biodegradation,2016,115:17-25.
16	Saito T,Okano K,Park H D,et al.Detection and sequencing of the microcystin LR-degrading gene,mlrA, from new bacteria isolated from Japanese lakes[J].FEMS Microbiology Letters,2003,229(2):271-276.
17	Imanishi S,Kato H,Mizuno M,et al.Bacterial degradation of microcystins and nodularin[J].Chemical Research in Toxicology,2005,18(3):591-598.
18	Okano K,Shimizu K,Kawauchi Y,et al.Characteristics of a microcystin-degrading bacterium under alkaline environmental conditions[J].Journal of Toxicology,2009,2009:954291.
19	Park H D,Sasaki Y,Maruyama T,et al.Degradation of the cyanobacterial hepatotoxin microcystin by a new bacterium isolated from a hypertrophic lake[J].Environmental Toxicology,2001,16(4):337-343.
20	Jiang Y,Shao J,Wu X,et al.Active and silent members in themlr gene cluster of a microcystin-degrading bacterium isolated from Lake Taihu, China[J].FEMS Microbiology Letters,2011,322(2):108-114.
21	Yan H,Wang H,Wang J,et al.Cloning and expression of the first gene for biodegrading microcystin LR bySphingopyxis sp. USTB-05[J].Journal of Environmental Sciences,2012,24(10):1816-1822.
22	Qin L,Zhang X,Chen X,et al.Isolation of a novel microcystin-degrading bacterium and the evolutionary origin ofmlr gene cluster[J].Toxins,2019,11(5):269-281.
23	Yang F,Zhou Y,Sun R,et al.Biodegradation of microcystin-LR and -RR by a novel microcystin-degrading bacterium isolated from Lake Taihu[J].Biodegradation,2014,25(3):447-457.
24	Chen J,Hu L B,Zhou W,et al.Degradation of microcystin-LR and RR by aStenotrophomonas sp. strain EMS isolated from Lake Taihu, China[J].International Journal of Molecular Sciences,2010,11(3):896-911.
25	Wang M,Casey P J.Protein prenylation: unique fats make their mark on biology[J].Nature Reviews Molecular Cell Biology,2016,17(2):110-122.
26	Ahearn I M,Haigis K,Bar-Sagi D,et al.Regulating the regulator: post-translational modification of RAS[J].Nature Reviews Molecular Cell Biology,2012,13(1):39-51.
27	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.
28	Dziga D,Wladyka B,Zielińska G,et al.Heterologous expression and characterisation of microcystinase[J].Toxicon,2012,59(5):578-586.
29	Dziga D,Lisznianska M,Wladyka B.Bioreactor study employing bacteria with enhanced activity toward cyanobacterial toxins microcystins[J].Toxins,2014,6(8):2379-2392.
30	Dexter J,Dziga D,Lv J,et al.Heterologous expression ofmlrA in a photoautotrophic host–engineering cyanobacteria to degrade microcystins[J].Environmental Pollution,2018,237:926-935.
31	Zilliges Y,Kehr J C,Mikkat S,et al.An extracellular glycoprotein is implicated in cell-cell contacts in the toxic cyanobacteriumMicrocystis aeruginosa PCC 7806[J].Journal of Bacteriology,2008,190(8):2871-2879.
32	Shimizu K,Maseda H,Okano K,et al.How microcystin-degrading bacteria express microcystin degradation activity[J].Lakes and Reservoirs: Research and Management,2011,16(3):169-178.
33	Hoefel D,Adriansen C M M,Bouyssou M A C,et al.Development of anmlrA gene-directed TaqMan PCR assay for quantitative assessment of microcystin-degrading bacteria within water treatment plant sand filter biofilms[J].Applied and Environmental Microbiology,2009,75(15):5167-5169.
34	Ho L,Hoefel D,Palazot S,et al.Investigations into the biodegradation of microcystin-LR in wastewaters[J].Journal of Hazardous Materials,2010,180(1/2/3):628-633.
35	Li J,Peng L,Li J,et al.Divergent responses of functional gene expression to various nutrient conditions during microcystin-LR biodegradation byNovosphingobium sp. THN1 strain[J].Bioresource Technology,2014,156:335-341.
36	Ho L,Tang T,Monis P T,et al.Biodegradation of multiple cyanobacterial metabolites in drinking water supplies[J].Chemosphere,2012,87(10):1149-1154.
37	Maghsoudi E,Fortin N,Greer C,et al.Cyanotoxin degradation activity andmlr gene expression profiles of aSphingopyxis sp. isolated from Lake Champlain, Canada[J].Environmental Science: Processes and Impacts,2016,18(11):1417-1426.
38	Yu N Y,Wagner J R,Laird M R,et al.PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes[J].Bioinformatics,2010,26(13):1608-1615.
39	Krogh A,È Larsson B,Von Heijne G,et al.Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[J].Journal of Molecular Biology,2001,305(3):567-580.
40	Yachdav G,Kloppmann E,Kajan L,et al.PredictProtein—an open resource for online prediction of protein structural and functional features[J].Nucleic Acids Research,2014,42(W1):W337-W343.
41	Xu Q,Fan J,Yan H,et al.Structural basis of microcystinase activity for biodegrading microcystin-LR[J].Chemosphere,2019,236:124281.
42	Dolence J M,Steward L E,Dolence E K,et al.Studies with recombinantSaccharomyces cerevisiae CaaX prenyl protease Rce1p[J].Biochemistry,2000,39(14):4096-4104.
43	Kjos M,Snipen L,Salehian Z,et al.The Abi proteins and their involvement in bacteriocin self-immunity[J].Journal of Bacteriology,2010,192(8):2068-2076.
44	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.
45	吴涓,钟升,王光云,等.一株降解微囊藻毒素菌种的鉴定及其活性研究[J].中国环境科学,2011,31(1):116-122.
	Wu J,Zhong S,Wang G Y,et al.Identification and activity of a bacterial strain for the biodegradation of microcystins[J].China Environmental Science,2011,31(1):116-122.
46	周洁,闫海,何宏胜,等.食酸戴尔福特菌USTB04生物降解微囊藻毒素的活性研究[J].科学技术与工程,2006,6(2):166-170.
	Zhou J,Yan H,He H S,et al.Activity ofDelftic acidovorans for the biodegradation of microcystins [J].Science Technology and Engineering,2006,6(2):166-170.
47	Ho L,Tang T,Monis P T,et al.Biodegradation of multiple cyanobacterial metabolites in drinking water supplies[J].Chemosphere,2012,87(10):1149-1154.
48	Wang X,Utsumi M,Gao Y,et al.Influences of metal ions on microcystin-LR degradation capacity and dynamics in microbial distribution of biofilm collected from water treatment plant nearby Kasumigaura Lake[J].Chemosphere,2016,147:230-238.
49	Feng L,Yan H,Wu Z,et al.Structure of a site-2 protease family intramembrane metalloprotease[J].Science,2007,318(5856):1608-1612.
50	Pryor E E,Horanyi P S,Clark K M,et al.Structure of the integral membrane protein CAAX protease Ste24p[J].Science,2013,339(6127):1600-1604.
51	Quigley A,Dong Y Y,Pike A C W,et al.The structural basis of ZMPSTE24-dependent laminopathies[J].Science,2013,339(6127):1604-1607.
52	Fujinaga M,Cherney M M,Oyama H,et al.The molecular structure and catalytic mechanism of a novel carboxyl peptidase fromScytalidium lignicolum[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(10):3364-3369.
53	Baker R P,Young K,Shi Y,et al.Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(20):8257-8262.
54	Ben-Shem A,Fass D,Bibi E.Structural basis for intramembrane proteolysis by rhomboid serine proteases[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(2):462-466.
55	Wang Y,Ha Y.Open-cap conformation of intramembrane protease GlpG[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(7):2098-2102.
56	Vinothkumar K R,Strisovsky K,Andreeva A,et al.The structural basis for catalysis and substrate specificity of a rhomboid protease[J].The EMBO Journal,2010,29(22):3797-3809.
57	Madeira F,Lee J,Buso N,et al.The EMBL-EBI search and sequence analysis tools APIs in 2019[J].Nucleic Acids Research,2019,47(W1):W636-W641.
58	United Nations Children s Fund (UNICEF),World Health Organization (WHO).Progress on household drinking water,sanitation and hygiene 2000-2017: special focus on inequalities[R].New York:UNICEF and WHO,2019:7-9.
59	Díez-Quijada L,Prieto A I,Guzmán-Guillén R,et al.Occurrence and toxicity of microcystin congeners other than MC-LR and MC-RR: a review[J].Food and Chemical Toxicology,2019,125:106-132.
60	Valeria A M,Ricardo E J,Stephan P,et al.Degradation of microcystin-RR bySphingomonas sp. CBA4 isolated from San Roque reservoir (Córdoba–Argentina)[J].Biodegradation,2006,17(5):447-455.
61	Yan H,Wang J,Chen J,et al.Characterization of the first step involved in enzymatic pathway for microcystin-RR biodegraded bySphingopyxis sp. USTB-05[J].Chemosphere,2012,87(1):12-18.
62	王俊峰.鞘氨醇单胞菌USTB-05降解微囊藻毒素-RR第一个基因的克隆与表达[D].北京:北京科技大学,2012:104-105.
	Wang J F.Cloning and expression of first gene involved in the biodegradation of microcystin-RR bySphingopyxis sp. USTB-05[D].Beijing:University of Science and Technology Beijing,2012:104-105.
63	闫海,王华生,刘晓璐,等.微囊藻毒素微生物降解途径与分子机制研究进展[J].环境科学,2014,35(3):1205-1214.
	Yan H,Wang H S,Liu X L,et al.Advances in the pathway and molecular mechanism for the biodegradation of microcystins[J].Environmental Science,2014,35(3):1205-1214.
64	Hashimoto E H,Kato H,Kawasaki Y,et al.Further investigation of microbial degradation of microcystin using the advanced Marfey method[J].Chemical Research in Toxicology,2009,22(2):391-398.
65	Manya H,Chiba A,Yoshida A,et al.Demonstration of mammalian proteinO-mannosyltransferase activity: coexpression of POMT1 and POMT2 required for enzymatic activity[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(2):500-505.
66	Harke M J,Steffen M M,Gobler C J,et al.A review of the global ecology, genomics, and biogeography of the toxic cyanobacterium,Microcystis spp.[J].Harmful Algae,2016,54:4-20.
67	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,Lü B,Li C.Advances in enzyme stability modification[J].CIESC Journal,2016,67(1):277-284.
68	赵道辉,彭春望,廖晨伊,等.面向生物能源的酶固定化的计算机模拟[J].化工学报,2014,65(5):1828-1834.
	Zhao D H,Peng C W,Liao C Y,et al.Computer simulation of bioenergy-oriented enzyme immobilization[J].CIESC Journal,2014,65(5):1828-1834.

[1]	程业品, 胡达清, 徐奕莎, 刘华彦, 卢晗锋, 崔国凯. 离子液体基低共熔溶剂在转化CO₂中的应用[J]. 化工学报, 2023, 74(9): 3640-3653.
[2]	孟令玎, 崇汝青, 孙菲雪, 孟子晖, 刘文芳. 改性聚乙烯膜和氧化硅固定化碳酸酐酶[J]. 化工学报, 2023, 74(8): 3472-3484.
[3]	陈雅鑫, 袁航, 刘冠章, 毛磊, 杨纯, 张瑞芳, 张光亚. 蛋白质纳米笼介导的酶自固定化研究进展[J]. 化工学报, 2023, 74(7): 2773-2782.
[4]	汤晓玲, 王嘉瑞, 朱玄烨, 郑仁朝. 基于Pickering乳液的卤醇脱卤酶催化合成手性环氧氯丙烷[J]. 化工学报, 2023, 74(7): 2926-2934.
[5]	毛磊, 刘冠章, 袁航, 张光亚. 可捕集CO₂的纳米碳酸酐酶粒子的高效制备及性能研究[J]. 化工学报, 2023, 74(6): 2589-2598.
[6]	张兰河, 赖青燚, 王铁铮, 关潇卓, 张明爽, 程欣, 徐小惠, 贾艳萍. H₂O₂对SBR脱氮效率和污泥性能的影响[J]. 化工学报, 2023, 74(5): 2186-2196.
[7]	刘瑞琪, 周栖桐, 张悦, 贺莹, 高静, 马丽. 基于金纳米颗粒修饰二氧化硅纳米花的生物传感器构建及应用[J]. 化工学报, 2023, 74(3): 1247-1259.
[8]	贾露凡, 王艺颖, 董钰漫, 李沁园, 谢鑫, 苑昊, 孟涛. 微流控双水相贴壁液滴流动强化酶促反应研究[J]. 化工学报, 2023, 74(3): 1239-1246.
[9]	苏伟怡, 丁佳慧, 李春利, 王洪海, 姜艳军. 酶促反应结晶研究进展[J]. 化工学报, 2023, 74(2): 617-629.
[10]	刘昕, 戈钧, 李春. 光驱动微生物杂合系统提高生物制造水平[J]. 化工学报, 2023, 74(1): 330-341.
[11]	胡阳, 孙彦. 酶分子的自驱动及其介导的微纳马达[J]. 化工学报, 2023, 74(1): 116-132.
[12]	谭卓涛, 齐思雨, 许梦蛟, 戴杰, 朱晨杰, 应汉杰. 辅酶自循环的氧化还原级联体系在生物催化过程中的应用：机遇与挑战[J]. 化工学报, 2023, 74(1): 45-59.
[13]	安绍杰, 许洪峰, 李思, 许远航, 李佳锡. 利用分子机器的组装与分解构建pH敏感性谷胱甘肽过氧化物人工酶[J]. 化工学报, 2022, 73(8): 3669-3678.
[14]	张劢, 田瑶, 郭之旗, 王叶, 窦广进, 宋浩. 光催化-生物杂合系统设计优化用于燃料和化学品绿色合成[J]. 化工学报, 2022, 73(7): 2774-2789.
[15]	孙甲琛, 孙文涛, 孙慧, 吕波, 李春. 甘草黄酮合酶Ⅱ催化甘草素特异性合成7，4′-二羟基黄酮[J]. 化工学报, 2022, 73(7): 3202-3211.