Insights into the mechanism of succinimide formation in an archaeal glutaminase

Succinimide (SNN), an intermediate formed during asparaginyl deamidation or aspartyl dehydration in proteins, is generally hydrolysis-prone, leading to isomerization to L/D α/β-aspartyl residue, with the latter being considered deleterious to protein structure and function. An unusually stable SNN-mediated conformational rigidity through restriction of the backbone dihedral angle, ψ, enhances the thermostability of glutamine amidotransferase (GATase) from Methanocaldococcus jannaschii (Mj). Although several structural features involved in maintaining a stable SNN and imparting SNN-mediated thermostability have been identified in MjGATase, the residues in the protein that catalyse the rapid and complete conversion of Asn109 to SNN remain unknown. Here, we investigated several site-directed mutants of MjGATase for their ability to retain Asn109 side chain in the unmodified form. Mass spectrometric analysis of 10 single mutants enabled the identification of residues that impacted the proportion of SNN and Asn population in the protein sample. This led to the generation of two double mutants that retained intact Asn109 side chain as observed in the mass spectra and crystal structures. These mutants with intact Asn residue at position 109, displayed lower thermal stability than the protein with the SNN modification. Further understanding of the deprotonation mechanism was addressed using QM/MM MD metadynamics simulations.