NMR-driven insights into the evolutionary adaptation and dynamic regulation of Mycobacterium tuberculosis adenylate kinase: The critical role of glycine-mediated flexibility.

Adenylate kinase (Adk) is essential for cellular energy homeostasis as it catalyzes the reversible transfer of γ-phosphate from ATP to AMP. In most long-variants, such as Escherichia coli Adk, large-scale domain motions, concerted movements of the AMP-binding domain (AMPbd) and ATP-lid, are essential for catalytic efficiency. However, the structural diversity observed in Adk variants, especially the short-variant Adk from Mycobacterium tuberculosis (MtAdk) featuring a truncated ATP-lid, raises questions regarding the adaptation of its dynamic regulation and functional mechanisms to support survival in resource-limited and hostile environments. Here, using 15N-chemical exchange saturation transfer experiments, we identified key glycine residues, G32 and G46 in AMPbd and G128 within the ATP-lid, that undergo slow conformational exchange on the millisecond timescale. Glycine substitutions with proline revealed their critical roles: G32P and G128P mutations significantly impaired catalytic turnover, whereas G46P exerted a moderate effect. Molecular dynamics simulations revealed that these substitutions restrict local flexibility; G32P and G128P lock the enzyme in an open, less active conformation, whereas G46P destabilizes the hinge region, thereby hindering proper domain closure. Our integrated approach reveals that these glycine-mediated flexibilities are essential for substrate recognition and catalysis in MtAdk. These findings highlight the fundamental mechanistic divergence between short- and long-variant Adks, emphasizing glycine-mediated conformational plasticity as key in enzyme regulation, with promising implications in targeted therapies against bacterial survival strategies.