Mass spectrometry based identification of <scp>AMP</scp> ‐O‐Tris generated by <i>Thermococcus onnurineus</i> Cas10

Cas10, the catalytic core of type III CRISPR-Csm systems, synthesizes cyclic oligoadenylate (cOA) second messengers to activate downstream immune responses. Although Cas10 activity is regulated by complex assembly, the nucleophile selectivity and off-pathway reactivity of isolated Cas10 remain poorly understood. Here, using HPLC separation and subsequent tandem mass spectrometry (MS/MS) analysis, we identify and structurally characterize AMP-O-Tris as a noncanonical adenylylated product generated by isolated Thermococcus onnurineus Cas10. Our results reveal that purified Cas10 exhibits relaxed nucleophile selectivity, diverting ATP turnover into nonproductive adenylylation involving buffer-derived nucleophiles. This suggests that effector complex assembly constrains Cas10 reactivity to promote efficient cOA synthesis and suppress off-pathway chemistry. Furthermore, interception of reactive intermediates by buffer-derived nucleophiles may represent a potential chemical fail-safe that limits unintended signaling when Cas10 is uncoupled from the complex. Together, our study provides mechanistic insight into Cas10 regulation and informs the development of robust type III-based diagnostic platforms. Impact statement Our study reveals that Cas10 exhibits latent catalytic flexibility when isolated, identifying a noncanonical adenylation reaction. These findings demonstrate how complex assembly constrains enzymatic specificity to prevent aberrant signaling. This mechanistic insight is crucial for improving the fidelity and design of next-generation CRISPR-based diagnostic platforms.