Biology subjects

Horiuchi, K.

Publications and source records attributed to Horiuchi, K..

3 recordsLinked to original sources

bioRxiv2026

Local and non-local impacts of intra-hexamer interactions on temperature compensation of KaiC

A hexameric clock protein KaiC exhibits a 24-hour phosphorylation cycle with a unique property termed temperature compensation. The period is kept constant over physiological temperatures through compensatory coordination of underlying elementary reactions. The temperature-compensated ATPase activity of KaiC is one such key reactions that potentially contribute to maintaining a constant circadian period. We identified four amino acid residues responsible for the temperature compensation in an N-terminal ATPase domain of KaiC. D82 and K172 were located in a primary site, and the ATPase activity of each alanine mutant showed a positive correlation with rising temperature. N62 and E69 constituted a secondary site, where each alanine replacement resulted in a negative correlation with the temperature. The primary site exerts a compensatory regulation over the ATPase cycle locally within the N-terminal domain. The secondary site prevents the ATPase activity from becoming over-compensated by suppressing another compensatory regulation mediated through a non-local interaction with a C-terminal domain of KaiC. Therefore, any imbalance between the local and non-local compensatory regulations in KaiC affects the temperature dependence of its phosphorylation rhythm.

biochemistry↗

bioRxiv2026

Identification of a Third Period-tuning Site in Cyanobacterial Clock Protein KaiC

KaiC, a clock protein in cyanobacteria, cycles between dephosphorylated and phosphorylated states in a 24-hour period in the presence of KaiA and KaiB. We identified the 322nd residue of KaiC as a third example of period-tuning sites. 322nd-site-directed saturation mutagenesis resulted in a variety of KaiC mutants exhibiting either shortened or lengthened cycles. The tunable range of the periods was from approximately 11 to 78 h without significantly compromising temperature compensation. We conducted biochemical analyses of the 322nd variants and examined their predicted structural models. In contrast to another known period-tuning site, where the period decreases sharply as the side-chain volume increases due to mutations, the cycle lengths correlate only modestly with bulkiness at the 322nd residues. The 322nd residue is located in a C-terminal domain of KaiC and influences ATPase cycles in both the C-terminal domain and an N-terminal domain through its interaction with a flexible loop connecting the two domains. The structural models predict that placing less bulky but polar side chains, such as serine and threonine, at the 322nd position leads to the formation of a hydrogen-bonding network between that site and the loop. This reduces the mobility of the loop, resulting in the longer cycles due to decreases in the ATPase activity of the N-terminal domain. Conversely, placing bulky residues such as phenylalanine at the 322nd position appears to alter the loop structure, shortening the periods by enhancing the ATP activities of both the domains. The third period-tuning mechanism is distinct from other known mechanisms. Significance StatementA Kai-protein clock system serves as a model for studying how long circadian rhythms are achieved. We identified the 322nd residue of KaiC as a third example of period-tuning sites that allow tuning of the period in either long- and short-period directions. The third period-tuning mechanism differs from the two previously known types in several respects. Previous studies have suggested that the ATPase activity in an N-terminal domain of KaiC is the primary regulator of the period. On the other hand, the 322nd residues of KaiC can affect the period by activating the ATPase cycle in its C-terminal domain. Our findings will stimulate future studies on the period-tuning mechanism mediated by the ATPase activity in the C-terminal domain of KaiC.

biochemistry↗

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↗