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Structural insights into the calcium dependence of Stig cyclases

机译：对Stig环化酶钙依赖性的结构见解

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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.
机译：来自Stigonematalean蓝细菌的Stig环化酶被归类为新型的钙依赖性环化酶，其催化罕见的反应级联，所述反应级联包括Cope重排，6-exo-trig环化和亲电子芳族取代。以前，我们在底物结合袋附近发现了两个钙离子。钙配位残基在所有Stig环化酶中都是保守的。在本研究中，我们使用定点诱变来研究钙协调作用。通过单独突变任一Ca〜（2+）结合位点与丙氨酸的配位残基，酶活性显着降低，这表明两个位点中Ca〜（2+）的存在对于催化至关重要。此外，其中Ca〜（2 +）-结合的N137被Ala取代的N137A的晶体结构显示出显着的局部构象变化，导致挤压的底物-结合袋使底物进入无效。总之，钙配位对于建立催化结构要素很重要。这些结果增加了对钙依赖性Stig环化酶作用机理的基本理解。

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