Abstract
The Stig cyclases from Stigonematalean cyanobacteria are classified as a novel type of calcium-dependent cyclases which catalyze an uncommon reaction cascade comprising Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution. Previously we found two calcium ions near the substrate-binding pocket. The calcium-coordinating residues are conserved in all Stig cyclases. In the present study, we use site-directed mutagenesis to investigate the role of calcium coordination. By individually mutating the coordinating residues in either of the Ca(2+)-binding sites to alanine, the enzyme activity is significantly reduced, suggesting that the presence of Ca(2+) in both sites is essential for catalysis. Furthermore, the crystal structure of N137A, in which the Ca(2+)-binding N137 is replaced by Ala, shows significant local conformational changes, resulting in a squeezed substrate-binding pocket that makes substrate entry ineffective. In conclusion, calcium coordination is important in setting up the structural elements for catalysis. These results add to the fundamental understanding of the mechanism of action of the calcium-dependent Stig cyclases.