Institute for Tuberculosis Research, University of Illinois at Chicago, Chicago, Illinois, USA.
Acta Crystallogr F Struct Biol Commun. 2020 Nov 1;76(Pt 11):524-535. doi: 10.1107/S2053230X20013370. Epub 2020 Oct 23.
The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P2, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, β = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower K and V than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.
来自重要病原体土拉弗朗西斯菌的 II 类果糖-1,6-二磷酸酶（FBPaseII）的晶体结构以 2.4 Å 的分辨率呈现。讨论了其与密切相关的分枝杆菌（MtFBPaseII）和大肠杆菌（EcFBPaseII）磷酸酶以及来自集胞藻 6803 的双磷酸酶的结构和功能关系。来自土拉弗朗西斯菌的 FBPaseII（FtFBPaseII）在一个单斜晶系晶体形式（空间群 P2，单元细胞参数 a = 76.30，b = 100.17，c = 92.02 Å，β = 90.003°）中结晶，在不对称单元中有四条链。链 A 在其活性中心有两个配位的 Mg 离子，这与以前的发现不同，并且推测它们的存在使其失活。该结构揭示了一个近似的 222（D）对称同源四聚体，类似于以前描述的 MtFBPaseII，它由晶体学二联体形成，并且与在晶体中 222 对称位置发现的精确四聚体不同。相反，近似同源四聚体与在集胞藻中的双磷酸酶非常相似，尽管在 FtFBPase 中没有发现别构效应物。FtFBPaseII 的活性位点的氨基酸序列和折叠导致结构特征与以前发表的 EcFBPaseII 更相似，而与 MtFBPaseII 不相似。天然 FtFBPaseII 的动力学参数与已发表的研究一致。对酶活性位点 Thr89Ser 和 Thr89Ala 突变的动力学分析与其他 II 类双磷酸酶的先前提出的机制一致。Thr89Ala 变体酶无活性，但 Thr89Ser 变体酶部分有活性，与天然酶相比，K 和 V 约低四倍。来自 FtFBPaseII 的结构和功能见解将为特定抑制剂的设计提供有价值的信息。
