Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without "damping," or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity. KaiC's two major phosphorylation sites, Ser-431 and Thr-432, become phosphorylated and dephosphorylated by interacting with KaiA and KaiB, respectively. Here, we mutate Thr-432 into Ser to find the oscillatory phosphoryl transfer reaction damps. Previously, this mutant KaiC was reported to be arrhythmic in vivo. However, we found that the mutant KaiC gradually loses the ability to run in an autonomous manner and stays constitutively phosphorylated after 3 cycles in vitro.
© 2023 The Authors. Published by American Chemical Society.