When the wrong metal stops the cycle: Dynamics and specificity in plant copper-dependent peptide cyclases.

Herein, we report the expression of split BURP domain peptide cyclases (BpCs), primarily CamB1 from Ceanothus americanus, in Escherichia coli using the pET22b vector without a fusion partner while retaining their disordered N-terminal region. To our knowledge, this represents the first full-length split BpC expressed and isolated without reliance on a stabilizing fusion tag (e.g., maltose binding protein, MBP). Both CamB1 and ArbB2, from Coffea arabica, were purified and refolded from inclusion bodies, and displayed robust catalytic turnover on a minimal peptide substrate. Copper titrations revealed that catalytic assays using glutathione as the reducing agent require copper far in excess of the stoichiometric number of active sites, with activity plateauing at ∼50-fold excess, likely reflecting competition with nonspecific copper binding or solution speciation. Using ascorbic acid in place of glutathione not only restores but also increases maximal activity, requiring only near-stochiometric copper. Metal impact studies demonstrated that noncognate metals inhibit activity. Zn(II) most severely inhibited BpC function at low micromolar concentrations in enzyme-initiated assays containing Cu(II), Zn(II), and glutathione, but this effect was markedly alleviated in reducing agent-initiated assays and instead resembled the modest inhibition by Ag(I), which fully suppressed activity only near 1 mM. These results highlight how assay order influences metal competition at the active site. Given that BURP-domain proteins are implicated in plant stress responses, including tolerance to metal exposure, these findings suggest that Zn(II) and Ag(I) inhibition may represent a biochemical mechanism by which environmental metal stress modulates BpC activity.