Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.
Biochimie. 2012 Mar;94(3):879-90. doi: 10.1016/j.biochi.2011.12.007. Epub 2011 Dec 16.
Inositol monophosphatase (IMPase) family of proteins are Mg(2+) activated Li(+) inhibited class of ubiquitous enzymes with promiscuous substrate specificity. Herein, the molecular basis of IMPase substrate specificity is delineated by comparative crystal structural analysis of a Staphylococcal dual specific IMPase/NADP(H) phosphatase (SaIMPase - I) with other IMPases of different substrate compatibility, empowered by in silico docking and Escherichia coli SuhB mutagenesis analysis. Unlike its eubacterial and eukaryotic NADP(H) non-hydrolyzing counterparts, the composite structure of SaIMPase - I active site pocket exhibits high structural resemblance with archaeal NADP(H) hydrolyzing dual specific IMPase/FBPase. The large and shallow SaIMPase - I active site cleft efficiently accommodate large incoming substrates like NADP(H), and therefore, justifies the eminent NADP(H) phosphatase activity of SaIMPase - I. Compared to other NADP(H) non-hydrolyzing IMPases, the profound difference in active site topology as well as the unique NADP(H) recognition capability of SaIMPase - I stems from the differential length and orientation of a distant helix α4 (in human and bovine α5) and its preceding loop. We identified the length of α4 and its preceding loop as the most crucial factor that regulates IMPase substrate specificity by employing a size exclusion mechanism. Hence, in SaIMPase - I, the substrate promiscuity is a gain of function by trimming the length of α4 and its preceding loop, compared to other NADP(H) non-hydrolyzing IMPases. This study thus provides a biochemical - structural framework revealing the length and orientation of α4 and its preceding loop as the predisposing factor for the determination of IMPase substrate specificity.
肌醇单磷酸酶（IMPase）家族蛋白是一类普遍存在的镁（Mg2+）激活、锂（Li+）抑制的多功能酶，具有广泛的底物特异性。在此，通过对葡萄球菌双特异性 IMPase/NADP(H)磷酸酶（SaIMPase-I）与其他具有不同底物兼容性的 IMPase 的比较晶体结构分析，阐明了 IMPase 底物特异性的分子基础，这种分析方法借助了计算机对接和大肠杆菌 SuhB 突变分析。与它的原核生物和真核生物的非水解 NADP(H)对应物不同，SaIMPase-I 活性位点口袋的复合结构与古菌水解的 NADP(H)双特异性 IMPase/FBPase 具有高度相似的结构。SaIMPase-I 的大而浅的活性位点裂缝能够有效地容纳大的进入底物，如 NADP(H)，因此，证明了 SaIMPase-I 具有显著的 NADP(H)磷酸酶活性。与其他非水解 NADP(H)的 IMPase 相比，SaIMPase-I 活性位点拓扑结构的深刻差异以及其独特的 NADP(H)识别能力源于远距离α4 螺旋（在人源和牛源中为α5）及其前导环的不同长度和取向。我们通过排除机制确定了α4 和其前导环的长度是调节 IMPase 底物特异性的最关键因素。因此，与其他非水解 NADP(H)的 IMPase 相比，SaIMPase-I 的底物混杂性是通过修剪α4 和其前导环的长度而获得的功能增益。因此，该研究提供了一个生化-结构框架，揭示了α4 和其前导环的长度和取向作为决定 IMPase 底物特异性的倾向因素。
