Dual role for Zn²⁺ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease

Abstract

We describe two uncommon roles for Zn²⁺ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg²⁺. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg²⁺, Mn²⁺, or Ca²⁺. Among these metal ions, Mg²⁺ and Mn²⁺ induce promiscuous cleavage by the enzyme, whereas Ca²⁺-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg²⁺- and Mn²⁺-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.