为了探究组蛋白赖氨酸特异性脱甲基酶的酶学性质,克隆并在大肠杆菌BL21(DE3)异源表达了斑马鱼来源的组蛋白赖氨酸脱甲基酶1(histone lysine specific demethylase 1,Lsd1)和咖啡来源的组蛋白赖氨酸脱甲基酶(jumonji C,Jmjc)基因,进一步优化了发酵产酶条件。利用镍柱亲和层析纯化表达产物,考察了重组酶的酶学性质。结果表明,成功构建2个来源的重组大肠杆菌菌株BL21(DE3)/pET28a-lsD1和BL21(DE3)/pET28a-jmjC,在发酵温度25 ℃、诱导剂异丙基-β-D-硫代半乳糖苷浓度0.1 mmol/L条件下获得了可溶性目的蛋白,并在咪唑浓度分别为100、200 mmol/L的条件下纯化洗脱得到纯酶。纯化的Lsd1、Jmjc最适反应温度分别为35、20 ℃,最适反应pH分别为7.0、8.0,Lsd1的温度稳定性相对较高,Jmjc的酸碱耐受力更强;在金属离子影响方面,Mn2+对Lsd1和Jmjc的酶活力都有明显的促进作用, Co2+、Mg2+、Ca2+、Zn2+、Ni2+对两者有不同程度的抑制作用;动力学研究发现,需要较长时间才能与底物反应,Lsd1和Jmjc的催化效率较低,kcat/Km分别为8.23×10-6、1.06×10-5 L/(mol·s)。研究结果为组蛋白赖氨酸脱甲基酶在非组蛋白的N—CH3类化合物降解和体外抑制剂中的研究提供了基础。
To investigate the enzymatic properties of histone lysine demethylase, the demethylases originated from zebrafish(Lsd1) and coffee(Jmjc) were cloned and heterologously expressed in E. coli BL21(DE3), respectively. The fermentation process was further optimized, and the enzymatic properties were studied after the purification of the expressed products via affinity chromatography on a nickel column. Based on the results, two E. coli recombinants (BL21 (DE3)/pET28a-lsD1 and BL21(DE3)/pET28a-jmjC) were constructed successfully. The soluble target protein could be obtained under 25 ℃ and 0.1 mmol/L isopropyl-β-D-thiogalactopyranoside(IPTG) induction. These two enzymes could be further purified by eluting with 100 mmol/L and 200 mmol/L imidazole. In terms of reaction activity, the corresponding optimum temperature and pH for Lsd1 were 35 ℃ and 7.0, while for Jmjc were 20 ℃ and 8.0. Comparing Lsd1 with Jmjc in stability, Lsd1 showed better thermostability, but Jmjc was found to have better tolerance to the change of pH. The enzyme activity of these two demethylases was promoted by the addition of Mn2+. In contrast, Co2+, Mg2+, Ca2+, Zn2+ and Ni2+ would limit the activity on both enzymes to varies extent. According to the kinetic study, the catalytic coefficient of these two enzymes was relatively low since the overall reaction time was long. The kcat/Km of Lsd1 and Jmjc were 8.23×10-6 L/(mol·s) and 1.06×10-5 L/(mol·s), respectively. The current results could provide meaningful information for the application of histone lysine demethylase in the field of the degradation of non-histone N—CH3 compound as well as the research about the development of inhibitors in vitro.
[1] SHI Y J, LAN F, MATSON C, et al.Histone demethylation mediated by the nuclear amine oxidase homolog LSD1 [J].Cell, 2004, 119(7):941-953.
[2] CULHANE J C, COLE P A.LSD1 and the chemistry of histone demethylation [J].Current Opinion in Chemical Biology, 2007, 11(5):561-568.
[3] FORNERIS F, BINDA C, VANONI M A, et al.Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process [J].FEBS Letters, 2005, 579(10):2 203-2 207.
[4] BINDA C, MATTEVI A, EDMONDSON D E.Structure-function relationships in flavoenzyme-dependent amine oxidations:A comparison of polyamine oxidase and monoamine oxidase [J].The Journal of Biological Chemistry, 2002, 277(27):23 973-23 976.
[5] TSUKADA Y I, FANG J, ERDJUMENT-BROMAGE H, et al.Histone demethylation by a family of JmjC domain-containing proteins [J].Nature, 2006, 439(7 078):811-816.
[6] HOU H F, YU H T.Structural insights into histone lysine demethylation [J].Current Opinion in Structural Biology, 2010, 20(6):739-748.
[7] MOSAMMAPARAST N, SHI Y.Reversal of histone methylation:Biochemical and molecular mechanisms of histone demethylases[J].Annual Review of Biochemistry, 2010, 79:155-179.
[8] LEE M G, WYNDER C, COOCH N, et al.An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation [J].Nature, 2005, 437(7 057):432-435.
[9] LAURENT B, RUITU L, MURN J, et al.A specific LSD1/KDM1A isoform regulates neuronal differentiation through H3K9 demethylation [J].Molecular Cell, 2015, 57(6):957-970.
[10] NICHOLSON T B, CHEN T P.LSD1 demethylates histone and non-histone proteins [J].Epigenetics, 2009, 4(3):129-132.
[11] BURG J M, LINK J E, MORGAN B S, et al.KDM1 class flavin-dependent protein lysine demethylases [J].Peptide Science, 2015, 104(4):213-246.
[12] HUANG J, SENGUPTA R, ESPEJO A B, et al.p53 is regulated by the lysine demethylase LSD1 [J].Nature, 2007, 449(7 158):105-108.
[13] ZHANG J, YUAN B F, ZHANG F, et al.Cyclophosphamide perturbs cytosine methylation in jurkat-T cells through LSD1-mediated stabilization of DNMT1 protein [J].Chemical Research in Toxicology, 2011, 24(11):2 040-2 043.
[14] HUANG Y, STEWART T M, WU Y, et al.Novel oligoamine analogues inhibit lysine-specific demethylase 1 and induce reexpression of epigenetically silenced genes [J].Clinical Cancer Research, 2009, 15(23):7 217-7 228.
[15] HUANG Y, GREENE E, MURRAY STEWART T, et al.Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes [J].Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(19):8 023-8 028.
[16] KANOUNI T, SEVERIN C, CHO R W, et al.Discovery of CC-90011:A potent and selective reversible inhibitor of lysine specific demethylase 1 (LSD1) [J].Journal of Medicinal Chemistry, 2020, 63(23):14 522-14 529.
[17] LAURENT B, SHI Y.Expression, purification, and biochemical analysis of the LSD1/KDM1A histone demethylase[J].Methods in Enzymology, 2016, 573:241-259.
[18] 翁新春, 方薇, 陈祎斐.食品工业废水处理工艺及节能探究 [J].资源节约与环保, 2015 (2):63;65.
WENG X C, FANG W, CHEN Y F.Study on wastewater treatment technology and energy saving in food industry[J].Resources Economization & Environmental Protection, 2015 (2):63;65.
[19] 郑元锋. 食品工业废水的生化处理与回用技术 [J].食品界, 2020 (6):104-105.
ZHENG Y F.Biochemical treatment and reuse technology of food industry wastewater [J].Food Industry, 2020 (6):104-105.
[20] 刘梦佳. 食品工业废水处理技术综述 [J].建筑与预算, 2019(4):32-36.
LIU M J.Summary of wastewater treatment technology in food industry [J].Architecture and Budget, 2019(4):32-36.
[21] DASH S S, GUMMADI S N.Enhanced biodegradation of caffeine by Pseudomonas sp.using response surface methodology [J].Biochemical Engineering Journal, 2007, 36(3):288-293.
[22] KOKURA K, SUN L D, FANG J.In vitro histone demethylase assays [J].Methods in Molecular Biology (Clifton, N.J.), 2015, 1 288:109-122.
[23] KING O N F, LI X S, SAKURAI M, et al.Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors [J].PLoS One, 2010, 5(11):e015535.
[24] GOKULAKRISHNAN S, CHANDRARAJ K, GUMMADI S N.A preliminary study of caffeine degradation by Pseudomonas sp.GSC 1182 [J].International Journal of Food Microbiology, 2007, 113(3):346-350.
[25] TANG H Z, YAO Y X, WANG L J, et al.Genomic analysis of Pseudomonas putida:Genes in a genome island are crucial for nicotine degradation [J].Scientific Reports, 2012, 2:377.
[26] TANG H Z, YAO Y X, ZHANG D K, et al.A novel NADH-dependent and FAD-containing hydroxylase is crucial for nicotine degradation by Pseudomonas putida [J].Journal of Biological Chemistry, 2011, 286(45):39 179-39 187.
