Structural insights into θ-type carbonic anhydrases 3 and 4: Tuning the directionality of CO2 hydration in a diatom.
TL;DR
Structural and functional characterization of a novel CA, θ-CA3, from the diatom Phaeodactylum tricornutum, elucidating its physiological role and catalytic mechanism are reported, offering a new perspective on the molecular basis of carbon fixation in diatoms.
Abstract
Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3 -) 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 CO2-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 HCO3 - 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.