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

The recombinantly expressed protein indoleglycerol phosphate synthase from the hyperthermophilic bacterium Thermotoga maritima (tIGPS) was purified an d characterized with respect to oligomerization state, catalytic properties and thermostability. This enzyme from the biosynthetic pathway of tryptoph an is a monomer in solution. In contrast to IGPS from the hyperthermophilic archaeon Sulfolobus solfataricus, tIGPS shows high catalytic activity at r oom temperature and only weak product inhibition. In order to test the hypo thesis that salt bridges in a critical context contribute to the high therm ostability of tIGPS, two solvent-exposed salt bridges were selected, based on its three-dimensional structure, for individual disruption by site-direc ted mutagenesis. The first salt bridge fixes the N terminus to the core of the protein, and the second serves as a clamp between helices alpha(1) and alpha(8) which are widely separated in sequence but adjacent in the (beta a lpha)(8)-barrel. Kinetics of irreversible heat inactivation reveal that the salt bridge crosslinking helices alpha(1) and alpha(8) stabilizes tIGPS mo re strongly than that tethering the N terminus. (C) 1999 Academic Press.