Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain.
Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain.
复制标题
脱氮副球菌电子转移黄素蛋白的晶体结构：保守黄素结合域的结构和静电分析。
DOI：
10.1021/bi9820917
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Kim,JJ
中科院分区：
文献类型：
--
作者：
Roberts,DL;Salazar,D;Fulmer,JP;Frerman,FE;Kim,JJ
对脱氮副球藻电子转移黄素蛋白(ETF)的晶体结构进行了测定，在2.6°分辨率下，其ANR因子为19.3%。除了一个单环区外，整个折叠与人类酶的折叠完全相同。就像人类的结构一样，西普的结构。反硝化ETF由三个不同的结构域组成，其中两个由α亚基贡献，第三个由β亚基贡献。对结构的仔细分析表明，含有AMPI63的环在一定程度上导致了β蛋白与AMP结合的高特异性。利用人类和P的序列和结构。作为模型，已经对ETF家族的几个成员进行了详细的序列比对，包括推测的FixA和FixB蛋白的序列。从这一比对中明显看出，在ETF家族的所有成员中，位于FAD辅因子紧邻的残基是相同的，除了W3A1ETF蛋白中的丝氨酸和亮氨酸残基分别替换了人残基αT266和βY16。人与人之间的离子差异图谱。在结构上的反硝基转移ETF确定了两种蛋白质之间在静电上非常相似的表面，从而支持了先前人ETF与猪中链酰辅酶A脱氢酶(MCAD)之间的对接模型。人ORP之间电子转移反应的离子强度依赖性分析。反硝化ETF和MCAD表明，人的ETF在低离子强度(∼10 Mequiv)时发挥最佳作用，而P。在较高的离子强度(>；75 mequiv)下，反硝化ETF是更好的电子受体。这表明两种蛋白质的静电表面电位有很大的不同，这与蛋白质之间等电点的差异是一致的。人体和人体的静电势分析。反硝化ETF显示，TEP。去硝化股ETF的负电荷更大。这种过量的负电荷可能是导致两个ETF黄素蛋白之间氧化还原电位差异的原因，并为MCAD与两个ETF反应的相反离子强度依赖性提供了解释。此外，通过对先前描述的人−P.反硝化嵌合ETF蛋白模型的分析，有可能确定ETF的一个区域参与与ETF-泛醌氧化还原酶的对接，ETF-泛醌氧化还原酶是ETF的生理电子受体。
The crystal structure of electron transfer flavoprotein (ETF) fromParacoccus denitrificanswas determined and refined to anR-factor of 19.3% at 2.6 Å resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of theP. denitrificansETF is comprised of three distinct domains, two contributed by the α-subunit and the third from the β-subunit. Close analysis of the structure reveals that the loop containing βI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human andP. denitrificansenzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues αT266 and βY16, respectively. Mapping of ionic differences between the human andP. denitrificansETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human orP. denitrificansETF and MCAD demonstrates that the human ETF functions optimally at low (∼10 mequiv) ionic strength, whileP. denitrificansETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human andP. denitrificansETFs reveals that theP. denitrificansETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human−P. denitrificanschimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.