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

Michigan State University, East Lansing MI

Investigators

Abstract

Geiger MCB 9982536 The 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.