Elettra | XRD2 Drug resistance is a silent epidemic of the 21st century, with antimicrobialresistant infections contributing to an estimated 1.27 million deaths globally in 2019. Among these threats, Mycobacterium tuberculosis (Mtb)— the agent of Tuberculosis (TB)—stands out for its adaptability and persistence, causing 1.4 million deaths in 2021. The evolution of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB has rendered current treatments increasingly ineffective. A newly characterized deaminase from Mycobacterium smegmatis, Msd, selectively targets mutagenic azascaffold purines and pyrimidines. Unlike canonical cytosine deaminases (CDAs), Msd does not act on standard nucleobases but instead detoxifies triazine-derived compounds. Crystal structures revealed Msd adopts a dimeric assembly featuring the hallmark α/β/α CDA core and conserved HxE and CxxC motifs that coordinate a catalytic zinc ion, Fig. 1 (a). The reaction proceeds via zinc-mediated water activation, a mechanism shared with other nucleobase deaminases. Surprisingly, however, Msd consistently adopts an open active site conformation in crystal structures, an uncommon trait for deaminases, where solvent shielding is typically essential for catalytic efficiency. This suggests that additional structural elements may dynamically regulate access to the active site, and understanding this could provide a framework for designing selective mechanism-based inhibitors against the mycobacterial deaminase family. To explore this, molecular dynamics (MD) simulations revealed two gating loops—β2–α2 and α4–α5—coordinate to open and close over the active site, Figs. 1 (b,c). β2–α2 loop is a structurally conserved loop in the CDA family, however, the α4–α5 loop is a unique structural insertion seen only in Msd, Fig. 1 (a). Substrate binding shifts these loop populations toward the closed conformation, occluding the active site and enabling catalysis. Mutational analysis confirmed that deletions or substitutions in these loops reduce activity and stability, especially within the conserved di-proline motif in the α4– α5 loop, Fig. 1 (d). These results highlight that both loop length and residue identity govern gating efficiency. To assess structural consequences, crystal structures of alanine mutants of the di-proline motif, P122A, P123A, and ΔP123 were solved. Although the overall fold remained intact, the α4–α5 loop displayed increased flexibility in P122A (Fig. 1 e) and altered curvature in ΔP123 (Fig. 1 f). In ΔP123, P122 shifted into the P123 position, altering loop architecture. To validate that these structural changes impact loop dynamics, we performed MD simulations on the P122A mutant. Despite similar global RMSFs to the wild type, P122A showed frequent transitions between open, partially closed, and closed states, unlike native Msd which predominantly adopts the partially closed conformation in the absence of ligand. RMSD profile of the α4–α5 loop shows the increased flexibility of the loop in P122A mutant, Fig. 1 (g). This increased flexibility was also observed as higher B-factor occupancy for the loop in the crystal structure of P122A, Fig. 1 (h). Additional parameters such as free energy surface analysis showed that P122A primarily populates the open state, confirming loss of stable closure. The key residue P122, which in native Msd rotates and stacks against W95 to stabilize closure, Fig. 1 (i), was replaced with alanine, abolishing this hydrophobic interaction. Distance and interaction energy analysis confirmed this loss in P122A. These findings demonstrate that the di-proline motif in the α4–α5 loop confers conformational rigidity essential for catalysis. P122, in particular, anchors loop closure through stacking with W95 from the adjacent subunit, creating a deamination-competent pocket. Its mutation reduces closure frequency and catalytic efficiency by shifting the conformational equilibrium toward inactive open states. Through an integrated approach involving crystallography, mutagenesis, kinetic assays, and molecular dynamics simulations, we now reveal how evolutionarily acquired insertion elements in Msd serve as allosteric modulators that finetune catalytic activity. These insertions, located distally from the catalytic zinc center, play a central role in stabilizing a substrate-accessible closed state, a prerequisite for productive catalysis. Our work identifies active-site closure as a universal mechanism across CDAs, but reveals that the mechanism of gating, especially the presence and composition of loop insertions, is enzyme-specific. Comparison with structurally characterized CDAs such as yeast cytosine deaminase (yCD) and Nitrosomonas europaea guanine deaminase (NeGD) shows that the second gating element varies across enzymes. In Msd, the unique α4–α5 loop plays this role, functionally mirroring the C-terminal helices/loops in yCD and NeGD. These loops spatially occupy the same region in the closed conformations of all three enzymes, underlining a conserved gating principle despite structural divergence. Msd presents a model wherein gating loops, modular insertions beyond the catalytic core, regulate access to the active site through dynamic closure, enabling substrate specificity for mutagenic bases. Additionally, while most deaminases possess two proton shuttles to act on natural nucleobases, Msd contains only one, explaining its lack of activity on natural nucleobase and preference for more nucleophilic aza-scaffold substrates. These insights offer a framework for targeting Msd-like enzymes in mycobacteria to disrupt detoxification of mutagens, opening avenues for overcoming antibiotic resistance through rational drug design. Selective Structural Tuning Enables Mutagenic Base Deamination in Mycobacteria Elettra Highlights 2024–2025 70
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