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<p>The gene (SSO1457) encoding a <span>l</span>-glutamate dehydrogenase (GDH) homolog from the thermoacidophilic archaeon <i>Saccharolobus solfataricus</i> P2 was overexpressed in <i>Escherichia coli</i>. At a substrate concentration of 50&#xa0;mM, the enzyme (SSO1457) produced exhibited much higher specific activity toward <span>l</span>-norvaline than <span>l</span>-glutamate at temperatures between 55 and 75°C, whereas the enzyme showed higher activity for <span>l</span>-glutamate than <span>l</span>-norvaline at 85°C. The crystal structures of both NAD<sup>+</sup>/2-oxovalerate-bound and NAD<sup>+</sup>/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 <span>l</span>-norvaline and contributes to high specific activities for <span>l</span>-norvaline.</p>

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

  • 期刊论文

  • 发表日期：

    2025年7月24日

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  • Itsuki Okabe,
  • Masashi Hirano,
  • Taketo Ohmori,
  • Minako Segawa,
  • Kazunari Yoneda,
  • Toshihisa Ohshima,
  • Haruhiko Sakuraba

摘要

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.