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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 草酸是一种对几乎所有生物体都有毒性的化合物，是形成草酸杆菌的主要能源，这种细菌存在于包括人类在内的几种哺乳动物的肠道中。在这种微生物中，草酸在草酰-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，在这种折叠中，两个亚基以互锁的二聚体形式连接在一起，就像链的两个环[1]。事实上，这种折叠是CoA转移酶III类家族的所有酶的特征[2]，从yfdW基因编码的大肠杆菌中的甲酰-CoA转移酶同源基因[3]和丁甜菜碱-CoA：肉碱辅酶A转移酶的晶体结构中可以看出[4]。作为实验和理论相结合的努力的一部分，目的是(I)了解引起互锁二聚体结构的分子原理，(Ii)阐明位于活性部位内的构象可移动的四甘氨酸环(Gly258-Gly259-Gly260-Gly261)的作用[1]，我们感兴趣的是表征FRC二聚体的动力学性质，以及底物结合对它们的调节。在我们最初的研究中，我们的主要目标是为获得外部项目资金获得足够的初步数据，我们试图检查四甘氨酸环运动与脱辅酶的低频正常模式之间的关系，以及我们通过X射线结晶学观察到的几个FRC/底物和FRC/中间复合体[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[7]。然而，将配体结合的影响包括在这样的模型中是非常困难的，所以我们现在希望进行更多计算要求更高的简正模式分析[8]。特别是，我们的目标是使用CHARMM(版本c34b1或c33b2)软件包[10]中实现的DIMB(混合基对角化)方法[9]来计算FRC二聚体的大规模振动运动。考虑到该体系由16,000多个原子组成(即使溶剂环境是使用广义出生的溶剂化模型[11]建模的)，这种计算的内存要求对于在本地UF高性能计算设施上进行这项研究来说要求太高。鉴于我们当地资源的这些限制，我们通过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温度因素的比较来仔细评估[12]。底物结合和反应中间体对Trp48的四甘氨酸环和侧链的相关运动的影响将在这些研究中特别感兴趣，因为我们实验室最近的工作证明了这种色氨酸在高草酸条件下排除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，还将为更复杂的定向MD模拟提供平台[14]，以寻求探索四甘氨酸环在中介催化中的作用。后一组苛刻的计算实验将构成NSF未来在2008年提交的提案的基础。文献引用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.和Cambillau，C.(2003)J.Biol.化学。278,34582-34586。4.Stenmark，P.，GurmuD.和Nordlund，P.(2004年)生物化学43,13996-14003。5.Jonsson，S.，Ricagno，S.，Lindqvist，Y.和Richards，N.G.J.(2004)J.Biol。化学。279,36003-36012。6.Berthold，C.L.，Toyota，C.G.，Richards，N.G.J.和Lindqvist，Y.(2007)J.Biol。化学。接受出版。7.特米兹，N.A.，Meirovitch，E.和Bahar，I.(2004)蛋白质：结构。功能。生物信息素。57468-480。8.Tama，F.和Brooks，C.L.，III(2006)Annu。生物物理学牧师。生物醇。结构。35,115-133。9.Mouawad，L.和Perahia，D.(1993)生物聚合物33,599-611。10.Brooks，B.R.，Bruccoleri，R.E.，Olafson，B.D.，States，D.J.，Swminathan，S.和Karplus，M.(1983)J.化学。4,187-217。11.Bashford，D.和Case，D.A.(2000)Annu。菲斯牧师。化学。51,129-152。12.ISIN，B.，Doruker，P.和Bahar，I.(2002年)生物物理学。J.82,569-581.13.丰田，C.G.，Berthold，C.L.，Gruez，A.，Jonsson，S.，Lindqvist，Y.，Cambillau，C.和Richards，N.G.J.(2007)J.已提交供出版。14.Park S.和Schulten，K.(2004)J.太棒了。120,5946-5961。关于FRC结构和机制的工作得到了国家卫生研究院(DK61666)和瑞典研究理事会-自然科学和工程科学理事会的支持

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