Structural insights into the calcium dependence of Stig cyclases

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 Ca²⁺-binding sites to alanine, the enzyme activity is significantly reduced, suggesting that the presence of Ca²⁺ in both sites is essential for catalysis. Furthermore, the crystal structure of N137A, in which the Ca²⁺-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.

Fig. 1. Calcium-binding sites of Stig cyclases and structurally similar CBMs. (A) The coordinating residues to Ca1 and Ca2 in Stig cyclases (subfamily 1: FamC1, green; FilC1, cyan; HpiU5, magenta; and subfamily 2: FimC5, yellow; FisC, purple) are shown as thin sticks. The protein fold of FamC1 (gray cartoon), the metal ions (orange spheres), and a water molecule (red sphere) in the structure are also shown. (B) The overall protein structures are shown as cartoon models, and the bound calcium ions as yellow spheres. Polysaccharides bound to the CBM structures are shown as stick models. The corresponding PDB ID is shown below each structure. Ca1 and Ca2 of the FamC1 structure are labeled, and the Ca2-equivalent site in each CBM structure is indicated by an arrow.

Fig. 2. Enzyme activity comparison of FamC1 and the variants. Recombinant proteins of wild-type FamC1 and the variant were subjected to one-pot reactivity analysis to estimate the rate of cyclized product production. The amounts of product of the variants are each measured and presented as percentages of the wild type enzyme. Each protein was examined in triplicate and the average ± SD was calculated.

Fig. 3. Overall structure and Ca1-binding site of the N137A variant. (A) Crystal structure of the N137A variant is shown as a cartoon model. Both monomeric (left panel) and dimeric (right panel) organization of the protein are displayed. (B) Stereo-view of superimposed wild-type FamC1 (cyan) and N137A (green). Ca1-coordinating residues of the wild-type and the equivalent residues of N137A are shown as thin stick models; the bound ligand of CI-4 and calcium ions as thick stick and sphere models; and a water molecule which coordinates Ca1 in wild-type FamC1 as a cyan sphere. Blue dashed lines indicate the metal-ion coordinate bonds. Numbers in parentheses indicate distance (unit Å).

Fig. 4. Active site interaction network in the FamC1 structures. (A) Stereo-view of partial CI-4-interacting network in FamC1 structures. The 137 residue, L138, the D214-Y89 pair, and CI-4 in the N137 variant (green) and wild-type FamC1 (cyan) are displayed. The 2F_o − F_c electron density maps of these residues and ligands are contoured at 2.0σ and shown in the right panel. Y89 in two poses are indicated. (B) Stereo-view of superimposed structures of N137 and wild-type FamC1 is shown with the same color scheme and features as in (A).