Structural Studies of 1L-myo-inositol 1-Phosphate Synthase

葡萄糖- 6-磷酸通过1l -肌醇- 1-磷酸合成酶（MIP合成酶）转化为1l -肌醇- 1-磷酸（MIP）是所有真核生物从头合成肌醇的第一个固定和限速步骤。含有肌醇的分子作为膜组分和关键的第二信使信号转导物种的重要性使得这种酶的功能和调节对许多生物学上重要的细胞功能（包括增殖、神经刺激、分泌和收缩）很重要。与大多数利用NAD的酶不同，MIP合成酶使用辅因子NAD作为共底物，而是催化作用。这种转化被认为是通过四个不同的步骤发生的：氧化、烯醇化、分子内醛环化和还原，所有这些都发生在一个单一的活性位点，没有中间产物的解离。由于该酶的催化机制有许多问题尚不清楚，且MIP合酶的结构信息完全不存在，因此将采用单晶x射线晶体学来确定MIP合酶的结构。此外，为了充分表征机理的结构细节，将确定几种基于机理的抑制剂- mip合酶晶体结构。这些结构将充分定义酶的三维折叠，并充分描述这一重要催化过程的许多结构细节。该项目的主要目标是了解一种酶，在这种情况下是MIP合成酶，如何催化葡萄糖分子转化为一种完全不同的结构，即肌醇。这种复杂的转化发生在这种酶的一个活性位点内，而在此过程中明显形成的许多稳定中间体中的任何一个都没有解离。对这一过程的关键洞察将来自于MIP合成酶与几种分子抑制剂结合的原子分辨率结构的确定，这些分子抑制剂在结构上模仿了在这一过程中形成的中间体。有了这些结构，就有可能在分子上详细地描绘出这一过程的复杂机制。肌醇在几乎所有真核生物中都起着重要的生物学作用，从膜结构到细胞间信号传导。

GeigerMCB 9982536The conversion of glucose 6-phosphate to 1L-myo-inositol 1-phosphate (MIP) by 1L-myo-inositol 1-phosphate synthase (MIP synthase) is the first committed and rate limiting step in the de novo biosynthesis of inositol in all eukaryotes. The importance of inositol containing molecules both as membrane components and as critical second messenger signal transduction species make the function and regulation of this enzyme important for a host of biologically important cellular functions including proliferation, neuro-stimulation, secretion and contraction. MIP synthase uses the cofactor NAD not as a co-substrate, as is the case in most NAD-utilizing enzymes, but catalytically. This transformation is thought to occur via four distinct steps: an oxidation, enolization, intramolecular aldol cyclization, and a reduction, all of which happens in a single active site with no dissociation of intermediates. Since many issues regarding the catalytic mechanism of this enzyme are not understood and structural information regarding MIP synthase is completely nonexistent, the structure of MIP synthase will be determined using single crystal X-ray crystallography. In addition, to fully characterize the structural details of the mechanism, several mechanism-based inhibitor-MIP synthase crystal structures will be determined. These structures will fully define the three-dimensional fold of the enzyme and fully delineate many of the structural details of this important catalytic process.The major goal of this project is to understand how a single enzyme, in this case MIP synthase, can catalyze the transformation of a glucose molecule into a completely different structure, an inositol. This complex transformation occurs within a single active site of this enzyme without dissociation of any of the many stable intermediates that apparently form during the process. Critical insight into this process will come from the determination of atomic resolution structures of MIP synthase bound to several molecular inhibitors that structurally mimic the intermediates that form in this process. With these structures it will be possible to map in molecular detail the complex mechanism of this process. Inositols play important biological roles in virtually all eukaryotic organisms, from membrane structure to intercellular signaling.