Biology subjects

Onoue, Y.

Publications and source records attributed to Onoue, Y..

3 recordsLinked to original sources

bioRxiv2024

Evolutionary Origins of Self-sustained Kai Protein Circadian Oscillators

Light-dark cycles affect photosynthetic efficiency in autotrophic cyanobacteria; therefore, determining whether ancient cyanobacteria possessed a self-sustained circadian clock when oxygenic photosynthetic systems were established is an important issue in chronobiology. Here we examined the oscillation of the clock protein KaiC in modern cyanobacteria, as well as the function and structure of ancestral Kai proteins, to determine the evolutionary origin of the self-sustained Kai-protein oscillators. The results show that the oldest double-domain KaiC in ancestral bacteria lacked the factors functionally and structurally essential for rhythmicity. The ancestral Kai proteins had acquired these factors through molecular evolution that occurred around Global Oxidation and Snowball Earth events, and were eventually inherited as a self-sustained circadian oscillator by the most recent common ancestor of cyanobacteria capable of oxygenic photosynthesis. This autonomous Kai protein oscillator was further inherited by most freshwater and marine cyanobacteria present today as an autotrophic basis for time-optimal acquisition and consumption of energy from oxygenic photosynthesis.

biochemistry↗

bioRxiv2024

The priming phosphorylation of KaiC is activated by the release of its autokinase autoinhibition

KaiC, a cyanobacterial circadian clock protein with autokinase activity, catalyzes the dual phosphorylation of its own S431 and T432 residues in a circadian manner in the presence of KaiA and KaiB. Priming phosphorylation at T432 is a key step that promotes secondary phosphorylation at S431. Although KaiA binding is considered essential for KaiC phosphorylation, the mechanisms underlying the activation and inactivation of priming phosphorylation remain elusive. We found that the priming phosphorylation proceeds even in the absence of KaiA, but is autoinhibited within KaiC, which decreases the rate constant to 0.019 h-1. The autoinhibition of KaiC and the mechanism underlying the release from autoinhibition by KaiA were examined by KaiC structural analysis, and by classical molecular dynamics and quantum mechanics / molecular mechanics simulations. We found that the side chain of T432 adopts two rotamers in dephosphorylated KaiC, one of which places T432 in a position suitable for a nucleophilic attack on the terminal phosphate of adenosine triphosphate (ATP). However, the nucleophilicity of T432 was insufficient to overcome an energy barrier of approximately 22 kcal mol-1 because the catalytic function of a nearby base, E318, was self-suppressed by hydrogen bonding to positively charged R385. Biochemical assays of KaiC mutants showed that the autoinhibition of KaiC autokinase activity is attenuated by conferring T432 high nucleophilicity through the KaiA-assisted release of R385 from E318 to E352. During the circadian cycle, R385 switches interacting partners to inactivate/activate the autokinase function and to ensure the unidirectionality of the KaiC phosphorylation cycle. Significance StatementKaiC, a central player in the circadian clock system of cyanobacteria, undergoes an ordered phosphorylation cycle in the presence of KaiA and KaiB. To elucidate the mechanism underlying the rhythmic regulation of the KaiC autokinase, we performed structural analyses, computational simulations, and biochemical assays of KaiC and its mutants. The results indicate that KaiC is essentially an autoinhibited autokinase, and the autoinhibition of primary phosphorylation at its T432 residue is attenuated by conferring it high nucleophilicity against the terminal phosphate of adenosine triphosphate. KaiA contributes to releasing the autoinhibition of KaiC in a morning phase by switching the interacting partners of R385 from a catalytic glutamate E318 to E352, as well as ensuring unidirectionality of the KaiC phosphorylation cycle.

biochemistry↗

bioRxiv2022

Formation of multiple flagella caused by a mutation of the flagellar rotor protein FliM in Vibrio alginolyticus

The marine bacterium Vibrio alginolyticus forms only a single flagellum at the cell pole. In Vibrio, two proteins (GTPase FlhF and ATPase FlhG) regulate flagellar number at the cell pole. We previously isolated a mutant strain characterized as NMB155 that forms multiple flagella despite the absence of mutations in flhF and flhG. NMB155 also exhibited straight swimming without a directional change in flagellar rotation. Whole-genome sequencing of NMB155 identified an E9K mutation in FliM that is a component of the C-ring in the flagellar rotor. Mutations in FliM result in defects in flagellar formation (fla) and flagellar rotation (che or mot); however, there are few reports indicating that FliM mutations increase the number of flagella. Here, we determined that the E9K mutation confers the multi-flagellar phenotype and also the che phenotype. The co-expression of wild-type FliM and FliM-E9K indicated that they were competitive in regard to determining the flagellar number. It had been shown that the ATPase activity of FlhG corresponds to the flagellar number. We observed that the ATPase activity of FlhG was increased by the addition of FliM but not by the addition of FliM-E9K. This indicates that the N-terminal region of FliM that includes the E9 residue interacts with FlhG to increase its ATPase activity, and the E9K mutation may inhibit this interaction. We concluded that FliM downregulate FlhG activity to inhibit the formation of additional flagella. ImportanceThe flagellar rotor generates a driving force to rotate the flagellum and is not involved in controlling the number of flagella in Vibrio. However, we observed that the E9K mutation in the rotor protein FliM confers multiple flagella. Our findings reveal a novel regulatory mechanism controlling flagellar number.

microbiology↗