Structural insight into the unique substrate specificity of glutamate dehydrogenase from Saccharolobus solfataricus.

Abstract

The gene (SSO1457) encoding a L-glutamate dehydrogenase (GDH) homolog from the thermoacidophilic archaeon Saccharolobus solfataricus P2 was overexpressed in Escherichia coli. At a substrate concentration of 50 mM, the enzyme (SSO1457) produced exhibited much higher specific activity toward L-norvaline than L-glutamate at temperatures between 55 and 75°C, whereas the enzyme showed higher activity for L-glutamate than L-norvaline at 85°C. The crystal structures of both NAD⁺/2-oxovalerate-bound and NAD⁺/2-oxoglutarate-bound SSO1457 were determined. Comparison of the two structures showed that the positioning of the substrate molecules and the surrounding residues is nearly identical in the two complexes. In the 2-oxoglutarate-bound structure, the C5-carboxylate group of 2-oxoglutarate is hydrogen-bonded with the side chains of Lys72, Arg188, and Ser351, as observed in other GDHs. By contrast, in the 2-oxovalerate-bound structure, the C01, C02, and C03 atoms of 2-oxovalerate are anchored via hydrophobic interactions to the side chains of Met93 and Val348. Site-directed mutagenesis shows that the side chain of Met93 mainly mediates the reactivity of SSO1457 towards L-norvaline and contributes to high specific activities for L-norvaline.