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.
Highlights
Xueke Tang,‡af Jing Xue,‡bc Yunyun Yang,d Tzu-Ping Ko, e Chin-Yu Chen,b Longhai Dai,b Rey-Ting Guo, bf Yonghui Zhang*d and Chun-Chi Chen*b
Stig cyclases from Stigonematalean cyanobacteria are a new family of enzymes, which catalyze an unusual cascade of carbon–carbon bond forming reactions comprising three steps: Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution,[1,2,3] enabling the transformation of a geranylated indolenine to generate a variety of hapalindole-type indole alkaloids
To investigate the role of individual calcium ion in a Stig cyclase, the six FamC1 residues whose side chains contribute to Ca2+coordination were mutated to Ala and each variant's activity was examined by measuring the amounts of the nal product
Summary
To investigate the role of individual calcium ion in a Stig cyclase, the six FamC1 residues whose side chains contribute to Ca2+coordination were mutated to Ala and each variant's activity was examined by measuring the amounts of the nal product. These conformational changes lead to deviations of the b4 and b7 strands and the b6–b7 loop These structural elements are held in position by Ca1-coordination in FamC1, Fig. 3 Overall structure and Ca1-binding site of the N137A variant. These results provide a reasonable explanation for the lower activity in the Ca1-depleted variant
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