Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: Construction of a 16-residue ion-pair network at the subunit interface

The role of an 18-residue ion-pair network, that is present in the glutamat e dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been st udied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium T hermotoga maritima. These two GDHs are 55% identical in amino acid sequence , differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introd uced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T, m aritima glutamate dehydrogenase showed that all anticipated ion-pairs are f ormed and that a 16-residue ion-pair network is present. Enlargement of exi sting networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturat ion. However, combination of destabilizing single mutations in most cases r estored stability, indicating the need for balanced charges at subunit inte rfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T1 58E/N117R resulted in an enzyme with a 30 minutes longer half-life of inact ivation at 85 degrees C, a 3 degrees C higher temperature optimum for catal ysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis th at large ion-pair networks do indeed stabilize enzymes from hyperthermophil ic organisms. (C) 1999 Academic Press.