7D97

Crystal structure of N109P mutant of GATase subunit of Methanocaldococcus jannaschii GMP synthetase

Summary for 7D97

• Entry DOI: 10.2210/pdb7d97/pdb
• Descriptor: GMP synthase [glutamine-hydrolyzing] subunit A (2 entities in total)
• Functional Keywords: hyperthermostable glutamine amidotransferase, gatase_n109p, ligase
• Biological source: Methanocaldococcus jannaschii DSM 2661
• Total number of polymer chains: 4
• Total formula weight: 84137.05

Authors

Bellur, A.,Dongre, A.,Kavya, J.,Balaram, H. (deposition date: 2020-10-12, release date: 2021-08-04, Last modification date: 2023-11-29)

Primary citation

Dongre, A.V.,Das, S.,Bellur, A.,Kumar, S.,Chandrashekarmath, A.,Karmakar, T.,Balaram, P.,Balasubramanian, S.,Balaram, H.
Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation.
Biophys.J., 120:3732-3746, 2021

PubMed Abstract: Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase and, using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100°C, imparted by an unexpectedly stable succinimidyl residue at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side-chain carboxylate group of its succeeding residue Asp110, as well as through n → π interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding, which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of the succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

PubMed: 34302792
DOI: 10.1016/j.bpj.2021.07.014

Experimental method

X-RAY DIFFRACTION (1.89 Å)