Trans-interface coupling in the tetrameric enzyme underlies allosteric control in Escherichia coli biosynthetic...

Allosteric regulation in biosynthetic L-threonine deaminase (BTD) has long been attributed to effector-induced conformational changes. Here, we identify a trans-interface coupling mechanism that regulates the active site architecture of EcIlvA, the BTD homolog of Escherichia coli. Starting from a low-activity, regulatory-domain-truncated variant (EcIlvA_1-335, termed CDL) that retains only the catalytic domain and the native linker region, we combined computational screening with experimental selection and directed evolution to identify a variant (designated HSG) bearing three mutations in the linker region (H322L, S328L, G334L). These mutations restore enzymatic activity to approximately two-thirds of the full-length EcIlvA level. Size-exclusion chromatography experiments suggested that the truncated CDL variant is dimeric in solution, whereas the HSG mutations restore the tetrameric form of the full length native protein. High-resolution crystal structures of CDL and HSG reveal two distinct functional states. They demonstrate that substitutions at the monomer-monomer interfaces within the dimer-of-dimers tetrameric assembly trigger inter-subunit rotation, propagating conformational changes across subunits and remodeling interactions at distal dimer-dimer interfaces. This allosteric cascade stabilizes a tetrameric assembly that hosts an open, catalytically competent active site. Molecular dynamics simulations further reveal that tetramerization in this form enhances global structural rigidity while retaining essential flexibility in the substrate-binding loops. Collectively, our findings establish that trans-interface-coupling within the dimer-of-dimers architecture of the tetrameric enzyme acts as a key allosteric mechanism in EcIlvA, providing direct structural and dynamic evidence for quaternary structure-driven allostery in a classical metabolic enzyme.