Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 − ) and plays an essential role in carbon fixation in marine diatoms. Here we report the structural and functional characterization of a novel CA, θ‐CA3, from the diatom Phaeodactylum tricornutum , elucidating its physiological role and catalytic mechanism. AlphaFold prediction, sequence alignment, and metal analysis showed that θ‐CA3 is a dimeric enzyme, with each monomer composed of two zinc‐binding catalytic domains. High‐resolution X‐ray crystallographic structures of domain 2 of θ‐CA3 in the CO 2 ‐bound form revealed the detailed substrate binding pattern in the active site. Site‐directed mutagenesis showed that Asp49 and Arg117 in the active site are essential for catalysis. Notably, introducing a negative charge near the active‐site entrance resulted in a mutant enzyme with markedly increased activity under acidic pH, suggesting that electrostatic modulation of the active‐site environment regulates proton transfer and catalysis. Furthermore, we identified an HCO 3 − ion at the dimer interface that contributes to enzyme activation. Collectively, our findings provide fundamental structural insight into how the active‐site electrostatic charges and metal environment govern the catalytic efficiency of θ‐CA3, offering a new perspective on the molecular basis of carbon fixation in diatoms.
Negoro et al. (Tue,) studied this question.