Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3A Symonds Street, Auckland 1142, New Zealand; School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand.
Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3A Symonds Street, Auckland 1142, New Zealand; School of Chemical Sciences, University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand.
Biochim Biophys Acta Proteins Proteom. 2018 Feb;1866(2):264-274. doi: 10.1016/j.bbapap.2017.08.018. Epub 2017 Aug 26.
Phosphoribosyltransferases (PRTs) bind 5'-phospho-α-d-ribosyl-1'-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding mode in type I, II and III PRTs indicates that AnPRT does not bind PRPP, or nearby metals, in the same conformation as other PRTs. A structure with a stereoisomer of PRPP bound to AnPRT from Mycobacterium tuberculosis (Mtb) suggests a catalytic or post-catalytic state that links PRib movement to metal movement. Crystal structures of Mtb-AnPRT in complex with PRPP and with varying occupancies of the two metal binding sites, complemented by activity assay data, indicate that this type III PRT binds a single metal-coordinated species of PRPP, while an adjacent second metal site can be occupied due to a separate binding event. A series of compounds were synthesized that included a phosphonate group to probe PRPP binding site. Compounds containing a "bianthranilate"-like moiety are inhibitors with IC values of 10-60μM, and K values of 1.3-15μM. Structures of Mtb-AnPRT in complex with these compounds indicate that their phosphonate moieties are unable to mimic the binding modes of the PRib or pyrophosphate moieties of PRPP. The AnPRT structures presented herein indicated that PRPP binds a surface cleft and becomes enclosed due to re-positioning of two mobile loops.
磷酸核糖基转移酶（PRTs）结合 5'-磷酸-α-d-核糖-1'-焦磷酸（PRPP），并将其磷酸核糖基（PRib）转移到特定的亲核试剂上。邻氨基苯甲酸磷酸核糖基转移酶（AnPRT）是一种混杂的 PRT，可磷酸化邻氨基苯甲酸和替代底物，是唯一的 III 型 PRT 实例。比较 I 型、II 型和 III 型 PRTs 中的 PRPP 结合模式表明，AnPRT 与其他 PRTs 不同，它不以相同的构象结合 PRPP 或附近的金属。来自结核分枝杆菌（Mtb）的 AnPRT 与 PRPP 的立体异构体结合的结构表明，这种酶的催化或后催化状态将 PRib 的运动与金属的运动联系起来。与活性测定数据互补的 Mtb-AnPRT 与 PRPP 复合物和两个金属结合位点占有率不同的晶体结构表明，这种 III 型 PRT 结合单个金属配位的 PRPP 物种，而相邻的第二个金属位点可以由于单独的结合事件而被占据。合成了一系列包含膦酸基团的化合物来探测 PRPP 结合位点。含有“双邻氨基苯甲酸”类似部分的化合物是抑制剂，IC 值为 10-60μM，K 值为 1.3-15μM。与这些化合物结合的 Mtb-AnPRT 的结构表明，它们的膦酸部分无法模拟 PRPP 的 PRib 或焦磷酸部分的结合模式。本文提供的 AnPRT 结构表明，PRPP 结合到一个表面裂缝中，并由于两个可移动环的重新定位而被封闭。
