Structural characterizations of the Mycobacterium tuberculosis AdaA-AlkA (Rv1317c) bifunctional enzyme and identification of enprofylline as a candidate synergistic DNA-repair targeting agent through integrated computational and experimental approaches.

Mycobacterium tuberculosis (M. tuberculosis) encodes a unique bifunctional AdaA-AlkA enzyme (Rv1317c) that fuses an N-terminal methyltransferase domain with a C-terminal glycosylase-derived domain, an arrangement confined to Actinobacteria and absent in Escherichia coli, where the two functions reside on separate proteins. Multiple sequence alignment across six bacterial species confirmed strict conservation of the four Zn2+-coordinating cysteines in the AdaA domain and the catalytic aspartate in the AlkA domain, while identifying Actinobacteria-specific substitutions (Leu125→Gln329, Tyr222→Val425, Tyr273→Thr480) that are in agreement with the experimentally reported loss of glycosylase activity. In the absence of an experimental structure, AlphaFold structure prediction, validated against E. coli Ada (1U8B) and AlkA (1DIZ) as domain-specific structural references yielded a high-confidence model. A novel ∼1138 Å3 interdomain cavity was identified and targeted through multi-level virtual screening of ∼2700 approved drugs, identifying Enprofylline with a docking score of -8.33 kcal mol-1500 ns molecular dynamics across two independent simulation replicates indicated stable ligand retention (COM ∼0.54 nm), ∼27% backbone RMSD reduction, suppression of inter-domain anti-correlated motions, and a replicate-averaged MM/PBSA binding free energy of -22.25 kcal mol-1 (upper-bound estimate excluding conformational entropy). In vitro validation against M. tuberculosis H37Ra demonstrated antimycobacterial activity (MIC = 25 μg/mL) with no cytotoxicity against THP-1 macrophages (CC50 > 100 μg/mL, SI > 4), and borderline synergy with rifampicin (FICI = 0.5), supporting Enprofylline as a candidate repurposed agent targeting the DNA-repair pathway with potential to potentiate frontline TB therapy.