The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solvent-exposed salt bridges.

The recombinantly expressed protein indoleglycerol phosphate synthase from the hyperthermophilic bacterium Thermotoga maritima (tIGPS) was purified and characterized with respect to oligomerization state, catalytic properties and thermostability. This enzyme from the biosynthetic pathway of tryptophan is a monomer in solution. In contrast to IGPS from the hyperthermophilic archaeon Sulfolobus solfataricus, tIGPS shows high catalytic activity at room temperature and only weak product inhibition. In order to test the hypothesis that salt bridges in a critical context contribute to the high thermostability of tIGPS, two solvent-exposed salt bridges were selected, based on its three-dimensional structure, for individual disruption by site-directed mutagenesis. The first salt bridge fixes the N terminus to the core of the protein, and the second serves as a clamp between helices alpha1 and alpha8, which are widely separated in sequence but adjacent in the (betaalpha)8-barrel. Kinetics of irreversible heat inactivation reveal that the salt bridge crosslinking helices alpha1 and alpha8 stabilizes tIGPS more strongly than that tethering the N terminus.