Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24000
標題: 利用X-ray 繞射法解析轉位子TnMERI1汞抗性操作組轉錄調控因子MerR1結構與分析汞離子還原酶 (MerA) 及有機汞裂解酶(MerB1、MerB2、MerB3) 之交互作用
X-ray crystallographic study of transcription regulator MerR1 and interaction analysis between mercuric reductase (MerA) and three organomercurial lyases (MerB1、MerB2、MerB3) from mercury resistance transposon TnMERI1
作者: 鄒曉薇
Zou, Xiao-Wei
關鍵字: MerB1
轉錄調控因子
MerB2
MerB3
MerA
MerR1
mercury resistance transposon TnMERI1
汞離子還原酶有機汞裂解酶
出版社: 生物化學研究所
引用: Ansari, A.Z., Chael, M.L., and O''Halloran, T.V. (1992). Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87-89. Barkay, T., Miller, S.M., and Summers, A.O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev 27, 355-384. Begley, T.P., Walts, A.E., and Walsh, C.T. (1986). Bacterial organomercurial lyase: overproduction, isolation, and characterization. Biochemistry 25, 7186-7192. Benison, G.C., Di Lello, P., Shokes, J.E., Cosper, N.J., Scott, R.A., Legault, P., and Omichinski, J.G. (2004). A stable mercury-containing complex of the organomercurial lyase MerB: catalysis, product release, and direct transfer to MerA. Biochemistry 43, 8333-8345. Brown, N.L., Stoyanov, J.V., Kidd, S.P., and Hobman, J.L. (2003). The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145-163. Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C.E., O''Halloran, T.V., and Mondragon, A. (2003). Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383-1387. Foulkes, E.C. (2000). Transport of toxic heavy metals across cell membranes. Proc Soc Exp Biol Med 223, 234-240. Gochfeld, M. (2003). Cases of mercury exposure, bioavailability, and absorption. Ecotoxicol Environ Saf 56, 174-179. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580. Helmann, J.D., Ballard, B.T., and Walsh, C.T. (1990). The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer. Science 247, 946-948. Huang, C.C., Narita, M., Yamagata, T., and Endo, G. (1999a). Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1. Gene 239, 361-366. Huang, C.C., Narita, M., Yamagata, T., Itoh, Y., and Endo, G. (1999b). Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan. Gene 234, 361-369. Lafrance-Vanasse, J., Lefebvre, M., Di Lello, P., Sygusch, J., and Omichinski, J.G. (2009). Crystal structures of the organomercurial lyase MerB in its free and mercury-bound forms: insights into the mechanism of methylmercury degradation. J Biol Chem 284, 938-944. O''Halloran, T.V., Frantz, B., Shin, M.K., Ralston, D.M., and Wright, J.G. (1989). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell 56, 119-129. Omichinski, J.G. (2007). Biochemistry. Toward methylmercury bioremediation. Science 317, 205-206. Pitts, K.E., and Summers, A.O. (2002). The roles of thiols in the bacterial organomercurial lyase (MerB). Biochemistry 41, 10287-10296. Schiering, N., Kabsch, W., Moore, M.J., Distefano, M.D., Walsh, C.T., and Pai, E.F. (1991). Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607. Nature 352, 168-172. Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60-89. 林慧姿. 轉位子TnMERI1上之廣效性有機汞裂解酶之純化精製與功能分析. In 生命科學系所 (中興大學), pp. 32. 林慧姿 (2007). 轉位子TnMERI1上之廣效性有機汞裂解酶之純化精製與功能分析. In 生命科學系所 (中興大學), pp. 32. 陳靖怡 (2003). 轉位子TnMERI1之汞抗性操縱組轉錄調控因子之精製與特性解析. In 生命科學系 (國立中興大學), pp. 71. 陳靖怡 (2007). 轉位子TnMERI1汞抗性操縱組轉錄調控因子之分子特性分析. In 生命科學系所 (中興大學), pp. 45.
摘要: 有機汞是脂溶性化合物,能輕易通過細胞膜與胞內蛋白質的硫醇基形成穩定的鍵結,造成蛋白功能的喪失或改變;有機汞也能夠穿過血腦障壁,導致神經中毒。鑑於其極高之生物毒性,因此探討自然界中降低有機汞毒性的機制顯得格外重要。革蘭氏陰性菌的汞抗性系統帶有一個有機汞裂解酶 (MerB),而在曾受有機汞嚴重汙染的水俁灣所發現的革蘭氏陽性菌Bacillus megaterium MB1卻擁有三個有機汞裂解酶 (MerB1, MerB2, MerB3),雖然序列比對顯示三者皆與革蘭氏陰性菌的MerB有相當的同源性,但MerB3具有較MerB1、MerB2以及陰性菌MerB更廣的受質特異性,能降解多種不同結構的有機汞。這些有機汞裂解酶的基因表現係受到轉錄調控因子MerR1與 MerR2的嚴密調控,當環境中出現汞離子時,MerR1會活化其調控序列O/PmerR1下游的基因表現,轉錄出MerB3、MerT、MerE、MerP以及 MerA 。MerR2 則可結合至 O/PmerR2 並活化 merR2 、 merB2 和 merB1 的基因表現,但MerR2的誘導因子目前尚未明瞭。 在降解汞的過程中,有機汞裂解酶負責切斷碳汞鍵以形成汞離子,由於此無機離子汞對生物體依然具有毒性,為了不使汞離子釋出而影響細胞內其他蛋白的功能,因此推測有機汞裂解酶會直接將產物汞離子交予還原酶以催化汞蒸氣的形成。酵素動力學分析指出此革蘭氏陰性菌之MerB與MerA間可能存在受質傳遞 (substrate channeling) 的現象,顯示MerB與MerA之間可能有直接的交互作用,本研究利用分子篩管柱層析法以及Dynamic Light Scattering (DLS) 分析發現MerB1、MerB2、MerB3個別與MerA的交互作用並不明顯,但加入二價鋅離子後似乎可略為提高兩酵素複合體的穩定度。活性測試顯示MerB3與其他兩個有機汞裂解酶的受質特異性並不相同,利用homology modeling分析發現其結構與E. coli 之MerB相較多了許多結構未知的區域,推測可能與其較廣的受質特異性有關。 我已成功培養出調控因子MerR1的蛋白晶體,X-射線繞射分析顯示此晶體屬於Primitive tetragonal晶系,可能之空間群為 P41212,其繞射解析度約可達3.5 Å ,晶胞參數為 a = 94.0 Å 、 b = 94.0 Å 、 c = 380.0 Å。未來期望藉由晶體結構了解MerR1蛋白調控轉錄作用之機轉、並比較其與MerR家族其他蛋白的異同。
Organomercuric compounds are liposoluble that can easily penetrate cell membrane. By binding tightly to the thiol groups, these compounds alter protein function and are poisonous. They can also diffuse across the blood-brain barrier as potent neurotoxins. Due to the highly toxic nature of organomercury, the corresponding bioremediation pathways have received tremendous attention in recent years. A few Gram negative bacteria have plasmid-borne mercury detoxifying system which contains an organomercourial lyase (MerB) capable of cleaving carbon-mercury bonds of organomercoury to produce divalent mercury ion (Hg2+). Interestingly, the Gram positive bacterium Bacillius megaterium MB1, isolated from the highly mercury-polluted Minamata Bay (Japan), was found to harbor a mercury-resistant operon (mer operon) with three organomercourial lyases (MerB1, MerB2, MerB3). Although sequence analysis revealed that the three enzymes are highly homologous, they exhibit distinct substrate specificity towards organomercuric compounds. Among them, MerB3 confers the broadest spectrum of mercury resistance. The expression of mer genes are regulated by two regulatory proteins, MerR1 and MerR2. In presence of mercury ion, MerR1 can bind to O/PmerR1 and active the transcription of merB3, merA, merT, merE and merA genes. On the other hand, MerR2 can bind to O/PmerR2 and modulate the expression of merR2, merB2 and merB1. The physiological activator for MerR2 is to date unknown. In the process of organomercury degradation, organomercourial lyase catalyzes the protonolysis of C-Hg bond to yield Hg2+. Because the product Hg2+ remains highly toxic, therefore, it should be passed directly from the MerB active site to that of the MerA to minimize the potential damaging effects of Hg2+. To test if direct interaction exists between the lyase and reductase, we performed gel filtration and Dynamic Light Scattering (DLS) analyses. While the interaction between apoenzymes was not discernable, the addition of Zinc ion appeared to slightly enhance their interaction. However, additional experiments would be required to validate this observation. I also verified that the three organomercurial lyases of B. megaterium MB1 exhibit distinct substrate specificity profile. Comparing with MerB1 and MerB2, MerB3 can catalyze the degradation of various organomercurial compounds, including PMA, MMC and PHMB. Homology models of the three MerBs show that the structure of MerB3 may be very different from E. coli MerB by containing more flexible regions. Whether this difference explains the broadest substrate specificity remains to be determined. In addition, I have successfully produced crystals of the full-length MerR1. Preliminary X-ray diffraction analysis indicated that the MerR1 crystals belong to Primitive tetragonal space groups with unit cell parameters a = 94.0 Å, b = 94.0Å, c = 380.0 Å. Upon the determination of MerR1 structure, we shall understand how it performs Hg2+-dependent transcription activation as well as the structural similarity and differences among MerR family members.
URI: http://hdl.handle.net/11455/24000
其他識別: U0005-0408200918375900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0408200918375900
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