The long-term objective of this project is to understand how new functionality is acquired by the enzymes and how new biochemical pathways evolve from the need to create more specialized functions. We plan to realize this goal by creating the 'library' of structures pertinent to a selected enzymatic function. A collection of enzymes from primitive to more advanced organisms (Archaea to Eukarya) will be created that will allow us to extract conserved or noel structures and to correlate them with new emerging function. More specifically, we are interested in enzymes involved in the synthesis of myo-inositol. Myo-inositol is generated by the conversion of D-glucose-6- in enzymes involved in the synthesis of myo-inositol is generated by the conversion of D-glucose-6- phosphate to L-myo-inositol-1-phosphate (via inositol-1-phosphate synthase, IPS) followed by specific dephosphorylation via inositol monophosphate (IMPase). This group of enzymes in archaea is particularly interesting because it provides a simplified model of inositol processing. The immediate goal of this study will be to obtain critical insights into the structure and function of both groups of enzymes. Specifically, we plan (1) to characterize crystallographically the previously cloned and purified IPS from A. fulgidus that is typical of the smaller IPS enzymes from bacteria and archaea, has no known homologs among PDB deposits, and which, in particular, requires divalent metal ion for the aldolase step. A series of crystallographic experiments: Apo- IPS (in particular, requires divalent metal ion for the aldolase step. A series of crystallographic experiments: Apo-IPS (in the presence of EDTA), the complexes with Mn2+ and/or Zn2+, G-6-P (no NAD+) (no G-6-P), is expected to provide the details of the enzymatic reaction. (2) We will also determine structures of dual activity enzymes from hyperthermophiles that very specifically catalyze the hydrolysis of inositol-1-phosphate (inositol monophosphatase, IMPase activity)) and fructose-1,6-bisphosphatase, FBPase, activity) from A. fulgidus and Thermatoga maratima. Our structural analysis of M. jannaschii, IMPase (MJ0109) in the presence of various substrates/products in combination with inhibitory/activating metal ions has provided unique insights into this bifunctional enzyme as well as a framework for the proposed studies. These IMPase/FBPase proteins, which are weakly inhibited by Li+, will be compared and contrasted with the (3) structure of E. coli IMPase (also known as SuhB), whose structure will also be solved, that is strongly inhibited by Li+ and cannot hydrolyze FBP similarly to mammalian IMPase. (4) Once the crystal structures of all the enzymes are determined, we plan to conduct a series of crystallographic as well as biochemical studies to determine the details of the proposed catalytic mechanism for IMPS and IMPases. Additionally we will try to resolve the controversy concerning metal ion roles in IMPase activity and the mode of Li+ inhibition. The structural comparison of highly Li+- sensitive eukaryotic and E. coli IMPases to Li+-insensitive hyperthermophilic IMPases is expected to provide a testable hypothesis about the mode of Li+ inhibition and the structural features for the dual specificity of the archaeal homologues.
