| The mechanisms which archaea employ to deal with osmotic stress were evaluated. Three systems were investigated to explore this concept; the dynamics and uptake of osmolytes by Halobacterium NRC-1, the thermostability of Methanococcus jannaschii glutamine synthetase (GS), and the mechanism of inositol-1-phosphate synthase (IPS) from Archaeoglobus fulgidus.;It has been suggested that Halobacterium NRC-1, an aerobic halophile of the order Halobacteriales, utilizes the "salt-in strategy" exclusively to balance the high osmotic strength of its environment. Growth of Halobacterium NRC-1 under various salt concentrations and subsequent analysis of the internal organic small molecules and potassium concentrations has suggested that citrate, alpha-glutamate, and succinate are present in significant concentrations.;The effect of solutes on the thermostability of the metabolic enzyme glutamine synthase (GS) from Methanococcus jannaschii was measured and compared to solute effects on a bacterial mesophilic counterpart. None of the solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein so that it could be heated above Tm for long periods of time. For the archaeal GS, protein-protein interactions appeared to be the dominant factor in stabilizing the enzyme to prolonged incubation above T m.;Archaeoglobus fulgidus and other hyperthermophilic archaea accumulate di-myo-1,1'-inositol phosphate (DIP) when grow at supraoptimal temperature. Inositol-1-phoshate synthase (IPS) is responsible for committing cellular resources to the production of DIP. The crystal structure of this enzyme has recently been solved [Bog Stec, unpublished]. Potential critical residues in the catalytic mechanism were identified and mutants made where each residue was changed to an alanine (L257A, K274A, K306A, and D332A). All mutants rendered the enzyme inactive. To effectively study the mechanism, assays were developed to detect binding of G-6-P (the substrate) and binding of NAD+ (the cofactor). Furthermore, various one and two dimensional NMR techniques were used in attempts to identify and confirm the presence of the 5-keto-(D)-glucose-6-phosphate intermediate. Preliminary results suggest that the intermediate formed is the keto compound. |
