A la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein XyIR to non-natural effectors

To investigate the activation mechanism of the enhancer-binding protein XyI R encoded by the TOL plasmid of Pseudomonas putida mt-2, a combinatorial li brary was generated composed of shuffled N-terminal A domains of the homolo gous regulators DmpR, XyIR and TbuT, reassembled within the XyIR structure. When the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely di fferent distribution of electronegative groups (e.g. nitrotoluenes), protei n variants were found that displayed an expanded inducer range including th e new effectors. Although the phenotypes endowed with the corresponding cha nges were largely similar, the modifications involved different sites withi n the A domain. The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding. These results provide a rationale for evolving in vitro regulator s a la carte that are responsive to predetermined, natural or xenobiotic ch emical species.