中国科学院生态环境研究中心机构知识库
Advanced  
RCEES OpenIR  > 土壤环境科学实验室  > 学位论文
题名: 厌氧微生物砷甲基转移酶的基因克隆及作用机制研究
作者: 王培培
学位类别: 博士
答辩日期: 2014-11
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 朱永官
关键词: ; 甲基化 ; 硫酸盐还原细菌 ; 产甲烷古菌 ; 甲基转移酶,arsenic ; methylation ; sulfate reducing bacteria ; methanoarchaea ; methyltransferase
其他题名: Gene cloning and functional study of the arsenite methyltransferase from anaerobic microorganism
学位专业: 环境科学
中文摘要:       砷(arsenic, As)是自然界中分布广泛的有毒元素,也是当今世界首要控制的环境污染物之一。砷的甲基化主要是指在生物体内发生的无机砷转化为单甲基砷、二甲基砷和三甲 基砷等衍生物的过程。该过程能够引起砷的形态、毒性和迁移性等变化,是砷元素生物地球化学循环的重要组成部分,也是目前极受关注的一种生物解毒机制。
      湿地、沉积物等厌氧环境是常见的砷污染汇聚地和砷污染再次释放的源泉。有研究报道砷的甲基化在厌氧条件下比好氧条件下更易发生,而厌氧微生物是厌氧环境中 砷甲基化过程的主要驱动者。厌氧微生物的砷甲基化对于砷的生物地球化学循环有积极的促进作用,也为微生物修复砷污染提供了应用前景。目前砷的甲基化研究主 要集中在好氧微生物,而对厌氧微生物的研究较少。因此,研究厌氧微生物的砷甲基化机制显得尤为重要。
      本论文以厌氧环境中普遍存在的厌氧微生物—硫酸盐还原细菌和产甲烷古菌为研究对象,初步研究了厌氧微生物的砷甲基转移酶的作用机制以及生化功能特性,旨在 加深人们对厌氧微生物作用于砷的生物地球化学方面上的认识,为详细阐明厌氧微生物的砷甲基化机制奠定基础。论文的主要研究内容及结果如下:
      (1)硫酸盐还原细菌Clostridium sp. BXM中砷甲基转移酶的作用机制及其催化作用位点研究。Clostridium sp. BXM可以将无机砷甲基化为单甲基砷(monomethylarsenic, MMAs)和二甲基砷(dimethylarsenic, DMAs)衍生物。通过同源克隆的方法从该菌株中克隆了一个亚砷酸甲基转移酶(arsenite S-adenosyl-methionine methyltransferase, ArsM)基因CsarsM,该基因在对砷敏感的大肠杆菌中表达,不但提高了该大肠杆菌对无机三价砷(arsenite, iAs(III))的抗性,而且使大肠杆菌获得了甲基化和挥发无机砷的能力。在合适条件下,表达CsarsM的大肠杆菌挥发砷量可达培养基总砷的 1.4%。CsarsM基因编码的酶被纯化,在体外可将iAs(III)甲基化为MMAs、DMAs和三甲基砷氧化物(trimethylarsine oxide, TMAsO)。氨基酸定点突变研究表明,CsArsM酶的三个半胱氨酸(cysteine, Cys)位点,即Cys65、Cys153和Cys203,在砷的甲基化过程中起关键作用。它们的任一缺失会导致酶甲基化iAs(III)或者MMAs的 能力丧失。此外,CsArsM酶的Cys29/30和Cys262分别被突变后,都造成酶活力的下降。
      (2)产甲烷古菌Methanosarcina acetivorans C2A中砷甲基转移酶的作用机制研究。M. acetivorans C2A能将无机砷甲基化为MMAs,但没有DMAs和气态砷生成。从该古菌中克隆了一个arsM同源基因MaarsM。与对照相比,表达MaarsM基因 的大肠杆菌在高达100 μM的无机砷浓度下仍具有显著抗性。在合适条件下,表达MaarsM的大肠杆菌能将接近100%的无机砷甲基化,产物以DMAs为主;也能将培养基中约 0.4%的无机砷转化为挥发砷。纯化的MaArsM酶在体外合适条件下能将体系中95%以上的iAs(III)甲基化为MMAs、DMAs和TMAO。
      (3)古菌MaArsM酶的生化功能解析。氨基酸定点突变研究表明,古菌MaArsM酶有三个重要的半胱氨酸,即Cys62、Cys150和 Cys200,在砷甲基化过程中起关键作用,其中Cys62的突变会大大降低酶甲基化As(III)的活性,而Cys150或Cys200的突变造成酶甲 基化砷的作用完全丧失。其他的半胱氨酸中,Cys31和Cys194分别突变后,可使酶活力升高,而Cys24/25或者Cys30的突变对酶的催化作用 基本没有影响。在MaArsM酶的催化体系中添加适量亚硒酸钠(selenite, Se(IV)),对砷的甲基化有促进作用,而加入过量的Se(IV),又表现抑制作用。其他金属离子Fe(II)、Mn(II)、Co(II)和 Zn(II)对于MaArsM酶的催化反应没有明显影响。多种巯基类化合物可以作为MaArsM酶的辅因子,与谷胱甘肽相比,辅酶M和高半胱氨酸对甲基化 的作用效率更高。不同浓度的底物、酶或者辅因子均会不同程度影响甲基化反应的效率。
英文摘要:       Arsenic (As) is a widespread toxic element in the environment, and one of the most urgent contaminants in the modern world. Arsenic methylation mainly occurs in the organism, in which inorganic As can be transformed into mono/di/trimethylarsenic derivatives. This process can directly influence the species, toxicity and mobility of As. Thus it plays an important role in As biogeochemistry cycling and known as a hot.
      Anaerobic environments, such as wetlands or sediments, usually act as sinks or secondary sources of As pollution. It is reported that As methylation is preferred in anaerobic conditions compared with aerobic conditions. Anaerobic microorganisms are major contributors for As methylation in anaerobic ecosystems. Arsenic methylation by anaerobic microorganisms can promote the biogeochemical cycling of As, and provide a prospect for microbial remediation of As pollution as well. So far, studies of As methylation mainly focus on the aerobic microorganisms, but little is known for the anaerobes. Therefore, it is critical to study the mechanism of As methylation in anaerobic microorganisms.
      In this study, sulfate reducing bacteria (SRB) Clostridium sp. BXM and methanoarchaea M. acetivorans C2A, both of which are widespread in anaerobic environments, were chosen as subjects to study the mechanisms and biochemical characters of As(III) methyltransferases. The main aims of this thesis are to provide insights into the roles of anaerobic microorganism in As biogeochemistry and basis for the in-depth study of the mechanism of As methylation in anaerobes. The major contents and conclusions in this thesis are as follows:
      (i) The study of mechanism and active sites of As(III) methyltransferases from SRB isolate Clostridium sp. BXM. Clostridium sp. BXM was firstly demonstrated to have the ability of transforming inorganic As to monomethylarsenic (MMAs) and dimethylarsenic (DMAs) derivatives. One arsenite S-Adenosyl-Methionine methyltransferase (ArsM) gene, CsarsM was cloned from Clostridium sp. BXM. Heterologous expression of CsarsM in an As-sensitive strain of E. coli conferred its resistance to As and the ability of methylation and volatilization of As. Under proper conditions, E. coli with CsarsM expression can volatilize 1.4% of the total As. Methyltransferase CsArsM was purified from recombinant E. coli and catalyzed the formation of mono/di/trimethylarsenic from As(III) in vitro. By the site-directed mutagenesis studies, we confirmed that Cys65, Cys153, and Cys203 of CsArsM are required for As(III) methylation, any mutation of which abolished enzymatic activities in methylating As(III) or MMAs. In addition, the mutation of Cys29/30 or Cys262 weakened the activity of CsArsM.
      (ii) The mechanism of As(III) methyltransferase in methanoarchaea, Methanosarcina acetivorans C2A. It was demonstrated that M. acetivorans C2A has the ability to transform inorganic As to MMAs, but not DMAs or volatile As. One arsM gene homologue, MaarsM was cloned from M. acetivorans C2A. Heterologous expression of MaarsM in an As-sensitive strain of E. coli conferred its resistance to As when exposed to As(III) concentration up to 100 μM. Under proper conditions, E. coli with MaarsM expression can methylate 100% of the total As in the medium, with DMAs as dominant species, and volatilize about 0.4% of the total As into atmosphere. Methyltransferase MaArsM was purified and can methylate more than 95% of As(III) in the reaction system into mono/di/trimethylarsenic in vitro.
      (iii) The biochemical function analysis of MaArsM enzyme. By the site-directed mutagenesis, we confirmed that three cysteines (Cys62, Cys150, Cys200) are necessary for the enzymatic activity. The mutation of Cys62 deeply decreased As(III) methylation by MaArsM, while the mutaiton of Cys150 or Cys200 completely abolished the methylating ability of MaArsM. In addition, the mutation of Cys31 or Cys194 enhanced the activity of MaArsM, while Cys24/25 and Cys30 were not concerned with the activity of MaArsM in methylating As. Selenite [Se(IV)] at a relatively low concentration increased the level of As methylation by MaArsM in vitro, while excessive Se(IV) inhibited As methylation. Other metal ions [Fe(II), Mn(II), Co(II), Zn(II)] did not affect As methylation by MaArsM. Many sulfhydryl compounds can serve as the cofactor of MaArsM in As methylation. Compared with glutathione (GSH), CoM and homocysteine were more efficient for As methylation. The different concentration of substrates (As,enzyme, S-Adenosyl-Methionine or GSH) also affected the level of methylation to some degree.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34515
Appears in Collections:中澳联合土壤环境研究室_学位论文

Files in This Item:
File Name/ File Size Content Type Version Access License
王培培--厌氧微生物砷甲基转移酶的基因克隆及作用机制研究.pdf(6101KB)学位论文--限制开放 联系获取全文

Recommended Citation:
王培培. 厌氧微生物砷甲基转移酶的基因克隆及作用机制研究[D]. 北京. 中国科学院研究生院. 2014.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[王培培]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[王培培]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2018  中国科学院生态环境研究中心 - Feedback
Powered by CSpace