BIOLOGY AND BIOCHEMISTRY OF THE METABLISM OF GREENHOUSE GASES BY MICROBES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。脱卤Desulfitobacterium dehalogenans是一种厌氧微生物，它可以通过将氯芳烃底物的还原脱卤耦合到一个称为脱卤呼吸的电子传递链中来获取能量（1,2）。cpr基因簇是一个由8个基因组成的簇，当厌氧菌感觉到环境中的氯代芳香族化合物时，它就会被诱导，其中许多是环境污染物(3)。含有维生素B12和FeS簇的还原性脱卤酶（CprA）已被证明可以催化羟基-多氯联苯和其他氯芳烃的脱卤(4)。基于与全球无氧代谢调节因子FNR和FixK的序列相似性，研究人员提出CprA和其他cpr基因簇组分的表达受CprK控制(3)。CprK还与cAMP受体蛋白CRP具有序列相似性，这提示CprK可能是转录调节因子CRP- fnr超家族的一员。我的合作者Steve Ragsdale和他的实验室不仅致力于阐明D. dehalogenans的还原性脱卤酶（CprA）的催化机制，而且还致力于阐明这种微生物感知有毒氯芳烃并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，并证明该蛋白与氯化芳香结合后，激活了cpr基因簇（脱盐呼吸操纵子）的转录。他最近还发现，这种蛋白质只在厌氧条件下与DNA结合，并受到氧化还原调节，只有在还原时才有活性（未发表的结果，Pop & Ragsdale）。CRP- fnr转录调节因子超家族的一个众所周知的成员是CRP本身。该蛋白的变构调节已经得到了很好的研究，效应物介导的CRP构象改变的作用对其作为转录因子的功能至关重要(5)。另一方面，CprK利用独特的效应物3-氯-4-羟基苯乙酸酯（CHPA），一种与cAMP完全不同的效应物。因此，对CprK构象和活性的效应介导的变构控制的结构研究将允许评估独特的效应-效应域相互作用的结构基础，以及CprK-cpr启动子区域。此外，提出的结构研究将是第一个对毒性多氯联苯作出反应的转录调节蛋白的研究。最后，还将通过获得活性和非活性构象的结构来探索新发现的该蛋白的氧化还原调控，从而深入了解CprK的另一层调控。为了补充Ragsdale实验室积极的、正在进行的功能研究，提出了具有以下长期目标的CprK结构研究。我们计划阐明CprK氧化还原调控的结构细节。x射线晶体学技术和方法将用于确定以下结构，有氧条件下CprK的非活性构象和厌氧条件下CprK的活性构象。两种结构的比较将为了解CprK是如何受其氧化还原状态调节提供有价值的见解。我们计划阐明效应介导的CprK构象和活性变构控制的结构细节。CprK与cpr启动子区域之间以及效应域与效应域之间的相互作用有待探索。将采用x射线晶体学技术和方法来评价这两个区域相互作用的结构基础。具体来说，将确定以下结构，CprK与效应器结合，CprK与效应器和DNA结合。2004年夏季，作为内布拉斯加州大学林肯分校氧化还原生物学中心夏季研究项目的参与者，Anthony Krueger在厌氧条件下结晶了CprK的活性形式（Ragsdale博士和Ryter博士，共同导师）。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¿。这些晶体随后被重复制作，并由NWU 2004届毕业生、拉格斯代尔实验室的实验室技术员本·比尔（Ben Biehl）将其衍射极限提高到3¿。在2004年秋季，Biehl先生还成功地用CHPA结合结晶了CprK，并希望进一步改进结晶条件将产生衍射质量的晶体。CprK晶体已被送往斯坦福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该蛋白的Se-Met衍生物结晶，以便MAD相位可以用于结构测定。引用1。Utkin， I.， C. Woese等人（1994）。中华微生物学杂志，2014(4):444 - 444。2. 维格尔，张晓明，等（1999）。中国生物防治学报，30(4):444 - 444。3. H. Smidt， M. van Leest等人（2000）。中国生物医学工程学报（英文版），21(5):444 - 444。4. Krasotkina， J.， T. Walters等人（2001）。中国生物医学工程学报，21(4):444 - 444。5. 哈曼，J.G.（2001）。生物化学学报，2015(1):1-17。6. http://www.hamptonresearch.com/support/pdf101/CG101SDC

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