BIOLOGY AND BIOCHEMISTRY OF THE METABLISM OF GREENHOUSE GASES BY MICROBES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。脱卤硫杆菌是一种厌氧微生物，它可以通过将氯芳烃底物的还原脱卤化反应耦合到电子传递链上来获得能量，这个过程被称为脱卤素呼吸(1，2)。CPR基因簇是一个由8个基因组成的簇，当厌氧菌在其环境中检测到氯代芳香族化合物时，它被诱导出来，其中许多是环境污染物(3)。含有维生素B12和FeS簇的还原脱卤酶(CPRA)被证明催化羟基多氯联苯和其他氯代芳烃的脱卤化(4)。CPRA和CPR基因簇的其他成分的表达被认为是由CPRK控制的，因为它与厌氧代谢的全球调节因子FNR和FixK序列相似(3)。CPRK还与cAMP受体蛋白CRP序列相似，提示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脱卤假单胞菌还原脱卤酶(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素呼吸的机制。Ragsdale实验室的研究生Stelian Pop已经对CPRK进行了表征，并证明了这种蛋白质在与氯化芳香族结合后，激活了CPR基因簇的转录，即脱卤素呼吸操纵子。他最近还发现，这种蛋白质只在厌氧条件下与DNA结合，并且受氧化还原调节，只有在还原时才活跃(未发表的结果，Pop&Ragsdale)。CRP-FNR超家族转录调控因子中的一个著名成员是CRP本身。这种蛋白的变构调节已经被很好地研究了，并且很好地理解了效应器介导的CRP构象变化对其作为转录因子的作用(5)。另一方面，CPRK使用了独特的效应器3-氯-4-羟基苯乙酸酯(CHPA)，这是一种与cAMP非常不同的效应器。因此，对CPRK构象和活性的效应器介导的变构调控的结构研究将有助于评估独特的效应器-效应域相互作用的结构基础，以及CPRK-CPR启动子区域。此外，拟议的结构研究将是对对有毒多氯联苯做出反应的转录调节蛋白的第一次研究。最后，新发现的该蛋白的氧化还原调节也将通过获得活性和非活性构象的结构来探索，这为深入了解CPRK的另一层调控提供了洞察力。为了补充Ragsdale实验室正在进行的积极的功能研究，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氧化还原调节的结构细节。为了确定CPRK在有氧条件下的非活性构象和在厌氧条件下的活性构象，将采用X射线结晶学技术和方法。对这两种结构的比较将为CPRK如何受其氧化还原状态的调控提供有价值的见解。我们计划阐明效应器介导的变构控制CPRK的构象和活性的结构细节。CPRK与CPR启动子区域的相互作用以及效应器与效应器结构域之间的相互作用都有待研究。将使用X射线结晶学技术和方法来评估这两个区域相互作用的结构基础。具体地说，将确定以下结构，CPRK与效应器结合，CPRK与效应器和DNA结合。在2004年夏天，Anthony Krueger作为内布拉斯加州大学林肯分校Redox生物学中心夏季研究项目(Ragsdale博士和Ryter博士的共同导师)的参与者，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中进行了表征。衍射图显示晶体确实是蛋白质，衍射率为4？随后，本·比尔重复了这些晶体，并将它们的衍射极限提高到3°。本·比尔是西北大学2004年毕业的学生，也是拉格斯代尔实验室的实验室技术人员。在2004年秋季，Biehl先生还成功地结晶了具有CHPA结合的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。比尔目前正致力于制造一种蛋白质的Se-Met衍生物来结晶，这样MAD相变就可以用于结构测定。参考文献1.Utkin，I.，C.Woese等人艾尔(1994年)。InJ系统细菌素44(4)：612-9。2.Wiegel，J.，X.Zhang，et.艾尔(1999年)。应用环境微生物65(4)：2217-21。3.Smidt，H.，M.van Leest，et.艾尔(2000年)。细菌素182(20)：5683-91。4.Krasotina，J.，T.Walters等人艾尔(2001年)。生物化学杂志276(44)：40991-7.5.哈曼，J.G.(2001)。生物物理学报1547(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