LARGE-SCALE MOTIONS IN THE INTERLOCKED ENZYME FORMYL-COA TRANSFERASE

联锁酶甲酰基-辅酶A转移酶中的大规模运动

• 批准号: 7956257
• 负责人: Nigel Gordon RICHARDS
• 金额: $ 0.08万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956257
• 关键词: Active Sites; Adopted; Bacteria; Binding; Biochemistry; Biomedical Research; Biopolymers; Carbon Dioxide; Carnitine; Catalysis; Characteristics; Code; Coenzyme A; Coenzyme A-Transferases; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer software; Data; Energy-Generating Resources; Engineering; Environment; Enzymes; Escherichia coli; Family; Florida; Formates; Frequencies; Funding; Future; Genes; Goals; Grant; High Performance Computing; Human; Institution; Laboratories; Ligand Binding; Link; Mediating; Memory; Methods; Modeling; Molecular; Motion; Organism; Orthologous Gene; Oxalates; Oxalobacter formigenes; Oxalyl-CoA decarboxylase; Property; Proteins; Publications; Reaction; Research; Research Personnel; Resources; Role; Science; Side; Solvents; Source; Structure; Supercomputing; System; Temperature; Time; Transferase; Tryptophan; United States National Institutes of Health; Universities; Work; X-Ray Crystallography; base; computer studies; dimer; formyl-coenzyme A transferase; gamma-butyrobetaine; glycyl-glycyl-glycyl-glycine; insight; interest; literature citation; microorganism; network models; programs; research study; simulation

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. Oxalate, a compound that is toxic to almost all organisms, is the primary energy source for Oxalobacter formigenes, a bacterium that is present in the gut of several mammalian species including humans. In this microorganism, oxalate is converted into formate and carbon dioxide in a catalytic cycle involving the enzymes oxalyl-CoA decarboxylase and formyl-CoA transferase (FRC). The latter enzyme is of special interest because it adopts a spectacular new fold in which the two subunits are linked together in an interlocked dimer, like two rings of a chain [1]. This fold, in fact, is characteristic for all enzymes in the Class III family of CoA transferases [2], as seen in the crystal structures of the formyl-CoA transferase ortholog in Escherichia coli coded by the yfdW gene [3], and butyrobetaine-CoA:carnitine CoA transferase [4]. As part of combined experimental and theoretical efforts to (i) understand the molecular principles that give rise to the interlocked dimer structure, and (ii) elucidate the role of a conformationally mobile tetraglycine loop (Gly258-Gly259-Gly260-Gly261) located within the active site [1], we are interested in characterizing the dynamical properties of the FRC dimer, and their modulation by substrate binding. In our initial studies, which have a key goal of obtaining sufficient preliminary data for acquiring external project funding, we seek to examine the relationship between the tetraglycine loop motions and the low-frequency normal modes of the apo-enzyme, and several FRC/substrate and FRC/intermediate complexes that we have observed by X-ray crystallography [5,6]. In preliminary experiments using the local computing facilities at the University of Florida (UF), we have explored the use of elastic network models, primarily because of their computational simplicity [7]. It is, however, very difficult to include the effects of ligand binding into such models and so we now wish to pursue more computationally demanding normal mode analyses [8]. In particular, we aim to calculate the large-scale vibrational motions of the FRC dimer using the DIMB (Diagonalization In a Mixed Basis) method [9] as implemented in the CHARMM (version c34b1 or c33b2) software package [10]. Given that this system is comprised of over 16,000 atoms (even when the solvent environment is modeled using a Generalized-Born solvation model [11]) the memory requirements of such a calculation are too demanding for performing this study on local UF high-performance computing facilities. Given these limitations in our local resources, we are requesting supercomputing time through the POPS program to perform these DIMB calculations. The results of these computational studies will be carefully evaluated by comparisons with crystallographic temperature factors [12]. The impact of substrate binding and reaction intermediates on the correlated motions of the tetraglycine loop and the side chain of Trp48 will be of particular interest in these studies as recent work in our laboratory has demonstrated the importance of this tryptophan in precluding the inhibition of FRC under conditions of high oxalate [13]. Not only will these calculations give new insights into the functional motions of the FRC dimer, they will also provide a platform for more sophisticated steered MD simulations [14] that will seek to explore the role of the tetraglycine loop in mediating catalysis. The latter set of demanding computational experiments will form the basis of a future NSF proposal submission in 2008. Literature citations 1. Ricagno, S., Jonsson, S., Richards, N., and Lindqvist, Y. (2003) EMBO J. 22, 3210-3219. 2. Heider, J. (2001) FEBS Lett. 509, 345-349. 3. Gruez, A., Roig-Zamboni, V., Valencia, C., Campanacci, V., and Cambillau, C. (2003) J. Biol. Chem. 278, 34582-34586. 4. Stenmark, P., Gurmu, D., and Nordlund, P. (2004) Biochemistry 43, 13996-14003. 5. Jonsson, S., Ricagno, S., Lindqvist, Y. and Richards, N. G. J. (2004) J. Biol. Chem. 279, 36003-36012. 6. Berthold, C.L., Toyota, C.G., Richards, N.G.J., and Lindqvist, Y. (2007) J. Biol. Chem. Accepted for publication. 7. Temiz, N.A., Meirovitch, E., and Bahar, I. (2004) Proteins: Struct. Funct. Bioinf. 57, 468-480. 8. Tama, F., and Brooks, C.L., III (2006) Annu. Rev. Biophys. Biomol. Struct. 35, 115-133. 9. Mouawad, L., and Perahia, D. (1993) Biopolymers 33, 599-611. 10. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., and Karplus, M. (1983) J. Comput. Chem. 4, 187-217. 11. Bashford, D., and Case, D.A. (2000) Annu. Rev. Phys. Chem. 51, 129-152. 12. Isin, B., Doruker, P., and Bahar, I. (2002) Biophys. J. 82, 569-581. 13. Toyota, C.G., Berthold, C.L., Gruez, A., Jonsson, S., Lindqvist, Y., Cambillau, C., and Richards, N.G.J. (2007) J. Bacteriol. Submitted for publication. 14. Park, S., and Schulten, K. (2004) J. Chem. Phys. 120, 5946-5961. Work on FRC structure and mechanism has been supported by the National Institutes of Health (DK61666) and the Swedish Research Council-Scientific Council for Natural and Engineering Sciences

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。 草酸盐是一种对几乎所有生物体都有毒的化合物，是产草酸杆菌的主要能量来源，产草酸杆菌是一种存在于包括人类在内的几种哺乳动物肠道中的细菌。在这种微生物中，草酸盐在涉及草酰辅酶A脱羧酶和甲酰辅酶A转移酶（FRC）的催化循环中转化为甲酸盐和二氧化碳。后一种酶特别令人感兴趣，因为它采用了一种壮观的新折叠，其中两个亚基以互锁的二聚体连接在一起，就像链的两个环[1]。事实上，这种折叠是CoA转移酶III类家族中所有酶的特征[2]，如在大肠杆菌中由yfdW基因编码的甲酰-CoA转移酶直系同源物[3]和丁酰甜菜碱-CoA：肉毒碱CoA转移酶[4]的晶体结构中所见。作为实验和理论结合努力的一部分，（i）理解产生互锁二聚体结构的分子原理，以及（ii）阐明位于活性位点内的构象移动的四甘氨酸环（Gly 258-Gly 259-Gly 260-Gly 261）的作用[1]，我们有兴趣表征FRC二聚体的动力学性质及其通过底物结合的调节。在我们的初步研究中，其关键目标是获得足够的初步数据以获得外部项目资金，我们试图检查四甘氨酸环运动与脱辅基酶的低频正常模式之间的关系，以及我们通过X射线晶体学观察到的几种FRC/底物和FRC/中间体复合物[5，6]。在使用佛罗里达大学（UF）本地计算设施的初步实验中，我们探索了弹性网络模型的使用，主要是因为它们的计算简单性[7]。然而，很难将配体结合的影响纳入此类模型中，因此我们现在希望追求计算要求更高的正态模式分析[8]。特别是，我们的目标是使用CHARMM（版本c34 b1或c33 b2）软件包[10]中实现的DIMB（混合基对角化）方法[9]计算FRC二聚体的大尺度振动运动。鉴于该系统由超过16，000个原子组成（即使使用广义玻恩溶剂化模型[11]对溶剂环境进行建模），这种计算的内存要求对于在本地UF高性能计算设施上执行本研究来说过于苛刻。考虑到我们本地资源的这些限制，我们正在通过POPS程序请求超级计算时间来执行这些DIMB计算。这些计算研究的结果将通过与晶体学温度因子的比较进行仔细评估[12]。底物结合和反应中间体对四甘氨酸环和Trp 48侧链的相关运动的影响将在这些研究中引起特别关注，因为我们实验室最近的工作已经证明了这种色氨酸在高草酸盐条件下排除FRC抑制的重要性[13]。这些计算不仅将为FRC二聚体的功能运动提供新的见解，还将为更复杂的操纵MD模拟提供平台[14]，该模拟将寻求探索四甘氨酸环在介导催化中的作用。后一组要求苛刻的计算实验将成为2008年NSF提案的基础。文献引用1. Ricagno，S.，琼森，S.，理查兹，N.，和Lindqvist，Y.等人（2003）EMBO J. 22，3210-3219。2. Heider，J.（2001）FEBS Lett. 509，345-349。3. Gruez，A.，Roig-Zamboni，V.，瓦伦西亚角，Campanacci，V.，和Cambillau，C.（2003）J.Biol.Chem.278，34582-34586。4. Stenmark，P.，Gurmu，D.，和Nordlund，P.（2004）Biochemistry 43，13996-14003。5.琼森，S.，Ricagno，S.，Lindqvist，Y.和理查兹，N. G. J.（2004）J.Biol.Chem.279，36003-36012。6. Berthold，C.L.，丰田汽车公司，理查兹，新泽西州，Lindqvist，Y.（2007）J. Biol. Chem. 7. Temiz，N.A.，Meirovitch，E.，巴哈尔岛05 The Dog of the Woman（2004）生物信息学57，468-480。8. Tama，F.，和布鲁克斯，C.L.，第三卷（2006年）生物物理学生物分子Struct. 35，115-133. 9.穆阿瓦德湖和Perahia，D.等人（1993）Biopolymers 33，599-611。10.布鲁克斯，B.R.，Bruccoleri，R.E.，Olafson，B. D.，美国，D. J.，Swaminathan，S.，Karplus，M.（1983）J. Comput. Chem.4，187-217。11. Bashford，D.，和Case，D.A.（2000）Annu. Rev. Phys. Chem. 51，129-152。12. Isin，B.，Doruker，P.，巴哈尔岛（2002）Biophys. J. 82，569-581. 13.丰田汽车公司，Berthold，C.L.，Gruez，A.，琼森，S.，Lindqvist，Y.，Cambillau，C.和理查兹，N. G. J.（2007）J. Bacteriol.已提交出版。14.公园，S.，和Schulten，K.等人（2004）J. Chem. Phys. 120，5946-5961。FRC结构和机制方面的工作得到了美国国立卫生研究院（DK 61666）和瑞典自然科学和工程科学研究委员会-科学理事会的支持