BIOLOGY AND BIOCHEMISTRY OF THE METABLISM OF GREENHOUSE GASES BY MICROBES

微生物代谢温室气体的生物学和生物化学

• 批准号: 7627625
• 负责人: JODI M RYTER
• 金额: $ 4.29万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627625
• 关键词: Aerobic; Allosteric Regulation; Anaerobic Bacteria; Aromatic Compounds; Binding; Biochemistry; Biology; Complement; Computer Retrieval of Information on Scientific Projects Database; Condition; Core Facility; Coupling; Crystallization; Cyclic AMP; Cyclic AMP Receptor Protein; DNA Binding; Data Collection; Desulfitobacterium; Electron Transport; Environment; Environmental Pollutants; Evaluation; Funding; Gene Cluster; Genetic Transcription; Goals; Grant; Hand; Harvest; Hydroxyl Radical; Institution; Laboratories; Mediating; Mentors; Metabolism; Methodology; Microbe; Molecular Conformation; Nebraska; Operon; Oxidation-Reduction; Participant; Pattern; Personal Satisfaction; Phase; Polychlorinated Biphenyls; Process; Promoter Regions; Proteins; Regulation; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Role; Source; Structure; Students; Suggestion; Techniques; Transcription Regulatory Protein; United States National Institutes of Health; Universities; Vitamin B 12; Work; base; conformer; cysteine rich protein; dehalogenation; falls; greenhouse gases; improved; insight; member; programs; structural biology; synchrotron radiation; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Desulfitobacterium dehalogenans is an anaerobic microbe that can harvest energy by coupling reductive dehalogenation of chloroaromatic substrates to an electron transport chain in a process called dehalorespiration (1, 2). The cpr gene cluster is an eight-gene cluster that is induced when anaerobes sense chlorinated aromatic compounds in their environment, many of which are environmental pollutants (3). The reductive dehalogenase (CprA), containing vitamin B12 and FeS clusters, has been shown to catalyze the dehalogenation of hydroxyl-PCB and other chloroaromatics (4). Expression of CprA and other components of the cpr gene cluster was proposed to be controlled by CprK based on its sequence similarity to FNR and FixK, global regulators of anaerobic metabolism (3). CprK also shares sequence similarity with cAMP receptor protein, CRP, leading to the suggestion that CprK might be a member of the CRP-FNR superfamily of transcriptional regulators. My collaborator Steve Ragsdale and his laboratory focus not only on elucidating the catalytic mechanism of the reductive dehalogenase (CprA) from D. dehalogenans, but also on elucidating the mechanism by which this microbe senses toxic chloroaromatics and regulates dehalorespiration. Stelian Pop, a graduate student in the Ragsdale lab, has characterized CprK and demonstrated that the protein, upon binding a chlorinated aromatic, activates transcription of the cpr gene cluster, the dehalorespiration operon. He has also recently discovered that this protein only binds DNA under anaerobic conditions and is redox regulated, active only when reduced (unpublished results, Pop & Ragsdale). A well known member of the CRP-FNR superfamily of transcriptional regulators is CRP itself. The allosteric regulation of this protein is well studied and the role of the effector-mediated CRP conformation changes central to its function as a transcription factor are well understood (5). CprK, on the other hand, utilizes the unique effector 3-chloro-4-hydroxyphenyacetate (CHPA), an effector quite unlike cAMP. Therefore, a structural study of the effector-mediated allosteric control of the conformation and activity of CprK would allow an evaluation of the structural basis of a unique effector-effector domain interaction, as well as the CprK-cpr promoter region. Additionally, the structural studies proposed would be the first study of a transcriptional regulatory protein that responds to toxic PCBs. Finally, the newly discovered redox regulation of this protein will also be explored by obtaining structures of both active and inactive conformers, offering insight into yet another layer of regulation of CprK. To complement the active, ongoing functional studies in the Ragsdale lab, structural studies of CprK, with the following long-term goals, are proposed. We plan to elucidate the structural details of the redox regulation of CprK. X-ray crystallographic techniques and methodologies will be employed in order to determine the following structures, The inactive conformation of CprK under aerobic conditions and, The active conformation of CprK under anaerobic conditions. Comparison of the two structures will provide valuable insight into how CprK is regulated by its redox state. We plan to elucidate the structural details of the effector-mediated allosteric control of the conformation and activity of CprK. Both the interaction between CprK and the cpr promoter region and between effector and the effector domain are to be explored. X-ray crystallographic techniques and methodologies will be employed to evaluate the structural basis of interactions in both regions. Specifically, the following structures will be determined, CprK with effector bound and, CprK with effector and DNA bound. During Summer 2004, Anthony Krueger crystallized the active form of CprK under anaerobic conditions as a participant in the University of Nebraska-Lincoln, Redox Biology Center Summer Research Program (Dr. Ragsdale and Dr. Ryter, co-mentors). These crystals were subsequently characterized at the Structural Biology Core Facility at UNL. The diffraction pattern revealed the crystals were indeed protein and diffracted to a resolution of 4 ¿. The crystals were subsequently repeated and their diffraction limit improved to 3 ¿ by Ben Biehl, a NWU 2004 graduate and lab technician in the Ragsdale lab. During Fall 2004, Mr. Biehl also successfully crystallized CprK with CHPA bound, and it is hoped that further refinement of the crystallization conditions will result in diffraction quality crystals. The CprK crystals have been sent to Stanford Synchrotron Radiation Laboratory for native data collection. Mr. Biehl is currently working on making a Se-Met derivative of the protein to crystallize so that MAD phasing may be utilized in structure determination. References 1. Utkin, I., C. Woese, et. al. (1994). Int J Syst Bacteriol 44(4): 612-9. 2. Wiegel, J., X. Zhang, et. al. (1999). Appl Environ Microbiol 65(4): 2217-21. 3. Smidt, H., M. van Leest, et. al. (2000). J Bacteriol 182(20): 5683-91. 4. Krasotkina, J., T. Walters, et. al. (2001). J Biol Chem 276(44): 40991-7. 5. Harman, J.G. (2001). Biochim Biophys Acta 1547(1): 1-17. 6. http://www.hamptonresearch.com/support/pdf101/CG101SDC.pdf

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 脱卤脱硫杆菌是一种厌氧微生物，可以通过在称为脱卤呼吸的过程中将氯代芳族底物的还原性脱卤与电子传递链偶联来收获能量（1，2）。 cpr基因簇是一个八基因簇，当厌氧菌感觉到环境中的氯代芳香族化合物（其中许多是环境污染物）时会被诱导。 含有维生素B12和FeS簇的还原脱卤酶（CprA）已被证明可催化羟基PCB和其他氯代芳烃的脱卤（4）。 基于CprK与FNR和FixK（厌氧代谢的全局调节剂）的序列相似性，提出CprA和cpr基因簇的其他组分的表达受CprK控制（3）。 CprK还与cAMP受体蛋白CRP具有序列相似性，这表明CprK可能是转录调节因子CRP-FNR超家族的成员。 我的合作者Steve Ragsdale和他的实验室不仅致力于阐明D.脱卤，而且还阐明了这种微生物感知有毒氯代芳烃和调节脱卤呼吸的机制。 Ragsdale实验室的研究生Stelian Pop对CprK进行了表征，并证明该蛋白质在结合氯代芳香族化合物后，会激活cpr基因簇（脱卤呼吸操纵子）的转录。 他最近还发现，这种蛋白质只在厌氧条件下结合DNA，并且是氧化还原调节的，只有在还原时才有活性（未发表的结果，Pop & Ragsdale）。 转录调节因子的CRP-FNR超家族的一个众所周知的成员是CRP本身。 该蛋白质的变构调节已得到充分研究，效应子介导的CRP构象变化对其作为转录因子的功能至关重要的作用也已得到充分理解（5）。 另一方面，CprK利用独特的效应物3-氯-4-羟基苯乙酸（CHPA），这是一种与cAMP非常不同的效应物。 因此，效应介导的变构控制的构象和活性的CprK的结构研究将允许一个独特的效应-效应域相互作用的结构基础的评估，以及CprK-CPR启动子区。 此外，提出的结构研究将是第一次研究的转录调节蛋白，响应有毒的多氯联苯。 最后，还将通过获得活性和非活性构象异构体的结构来探索新发现的这种蛋白质的氧化还原调节，从而深入了解CprK的另一层调节。 为了补充Ragsdale实验室正在进行的功能研究，提出了CprK的结构研究，并提出了以下长期目标。 我们计划阐明CprK的氧化还原调节的结构细节。 将采用X射线晶体学技术和方法来确定以下结构， 在有氧条件下CprK的非活性构象， 厌氧条件下CprK的活性构象。 这两种结构的比较将提供有价值的洞察力如何CprK是由其氧化还原状态调节。 我们计划阐明CprK的构象和活性的效应介导的变构控制的结构细节。 CprK和cpr启动子区域之间的相互作用以及效应器和效应器结构域之间的相互作用都有待探索。 X射线晶体学技术和方法将被用来评估在这两个地区的相互作用的结构基础。 具体地，将确定以下结构， 效应子结合的CprK， 具有效应子和DNA结合的CprK。 2004年夏季，Anthony Krueger作为内布拉斯加大学林肯分校氧化还原生物中心夏季研究计划的参与者（Ragsdale博士和Ryter博士，共同导师），在厌氧条件下结晶了CprK的活性形式。 这些晶体随后在UNL的结构生物学核心设施进行了表征。 衍射图显示晶体确实是蛋白质，衍射分辨率为4 º。 晶体随后被重复，其衍射极限提高到3 º，由本Biehl，西北大学2004年毕业生和实验室技术员在拉格斯代尔实验室。 在2004年秋季，Biehl先生还成功地结晶了与CHPA结合的CprK，并希望进一步细化结晶条件将产生衍射质量的晶体。 CprK晶体已被送往斯坦福大学同步辐射实验室进行本地数据收集。 Biehl先生目前正致力于使蛋白质的Se-Met衍生物结晶，以便在结构测定中使用MAD定相。 引用 1. 乌特金岛C. Woese，et.等（1994）。Int J Syst Bacteriol 44（4）：612-9. 2. Wiegel，J.，X. Zhang，et.等（1999）。Appl Environ Microbiol 65（4）：2217-21. 3. S.，H.，M.货车莱斯特等。（2000）。J Bacteriol 182（20）：5683-91. 4. Krasotkina，J.，T. Walters，et.（2001）。J Biol Chem 276（44）：40991-7. 5. Harman，J.G.（2001年）的第10页。Biochim Biophys Acta 1547（1）：1-17. 6. http://www.hamptonresearch.com/support/pdf101/CG101SDC.pdf