STRUCTURAL DETERMINATION OF QUEA ENZYMES

这个子项目是许多利用 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。 QueA酶催化Q前体环氧肌苷（oQ）的2，3-环氧-4，5-二羟基环戊烷环的形成。S-腺苷-L-甲硫氨酸（S-adenosyl-L-methionine，简称ADMet）与tRNA的7-氨甲基-7-脱氮鸟嘌呤（7-aminomethyl-7-deazaguanine）在第34位反应，生成腺嘌呤、甲硫氨酸和修饰的tRNA（oQ在第34位）。oQ的环氧-环戊烯二醇部分来源于C3 Met的核糖基部分，这是C3 Met在酶促反应中作为核糖基供体的化学计量使用的唯一已知实例。鸟苷{7-（4，5-顺式-二羟基-2-环戊烯-1-基）-氨基）-甲基）-7-脱氮鸟苷}是一种高度修饰的核苷，存在于天冬氨酸、天冬酰胺、酪氨酸和组氨酸tRNA的34位，即反密码子摆动位置。虽然肌苷存在于细菌和真核生物中，但它仅在细菌中从头合成。因此，真核生物从肠道植物群中获得作为膳食营养素的肌苷，并使用肌苷-tRNA核糖基转移酶将相应的碱基肌苷掺入tRNA中。 我们已经开始对野生型酶和GST（谷胱甘肽-S-转移酶）融合酶进行结晶尝试。 含有所需肽或目的蛋白质的融合蛋白的形成已成功地用于困难蛋白质的结晶。由于我们在野生型酶的结晶方面遇到的最初困难，我们开始了QueA作为GST融合蛋白的平行结晶尝试。 同样重要的是注意到GST融合蛋白显示出与野生型QueA酶相似的活性。 这将意味着活性位点和RNA结合位点不被GST标签阻断。因此，我们也在尝试QueA：GST蛋白与底物和辅因子的复合物的结晶。 初步结果表明，GST融合蛋白是一个更好的候选结晶。 我们有QueA-GST-融合蛋白的小晶体，其显示出约3.0 <$分辨率的衍射。

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. The QueA enzyme catalyzes the formation of the 2,3-epoxy-4,5-dihydroxycyclopentane ring of the Q precursor epoxyqueuosine (oQ). S-adenosyl-L-methionine (AdoMet) reacts with 7-aminomethyl-7-deazaguanine of tRNA at position 34 to yield adenine, methionine, and a modified tRNA with oQ at position 34. The epoxy-cyclopentenediol moiety of oQ originates from the ribosyl portion of AdoMet, which is the only known example of the stoichiometric use of AdoMet as a ribosyl donor in an enzymatic reaction. Queuosine {7-(((4,5-cis-dihydroxy-2-cyclopentene-1-yl)-amino)-methyl)-7-deazaguanosine} is a hypermodified nucleoside that occurs at position 34, the anticodon wobble position, of aspartate, asparagine, tyrosine, and histidine tRNAs. Although queuosine is found in bacteria and eukaryotes, it is synthesized de novo exclusively in bacteria. Therefore, eukaryotes obtain queuosine as a dietary nutrient and from intestinal flora, and incorporate the corresponding base queuine into tRNA using the enzyme queuine-tRNA ribosyltransferase. We have started crystallization attempts on the wild type enzyme and a GST (Glutathione-s-transferase)-fused enzyme. The formation of fusion proteins containing the desired peptide or protein of interest have been successfully used for the crystallization of difficult proteins. Because of the initial difficulty we had with the crystallization of the wild type enzyme, we started parallel crystallization attempt of QueA as a GST-fusion protein. It is also important to note that the GST-fusion protein showed similar activity to the wild type QueA enzyme. This would mean that the active site and the RNA binding site are not blocked by the GST tag. Therefore, we are also attempting the crystallization of the complexes of the QueA:GST protein with substrate and cofactor. The initial results show that the GST-fusion protein is a better candidate for crystallization. We have small crystals of the QueA-GST-fusion protein which showed diffraction to around 3.0 ¿ resolution.