Observation of a calcium-binding site in the γ-class carbonic anhydrase from Pyrococcus horikoshii

Carbonic anhydrases are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbon­ate. Three crystal structures of γ-class carbonic anhydrase (one of which is bound to a bicarbonate molecule) from the aerobic OT3 strain of the hyperthermophilic archeon Pyrococcus horikoshii have been solved by molecular replacement in space group F4₁32. The asymmetric unit contains a monomer of 173 amino acids and a catalytic Zn²⁺ ion. The protein fold is a regular prism formed by a left-handed β-helix, similar to previously reported structures. The active-site Zn²⁺ ion located at the interface between the two monomers is bound to three histidyl residues and a water molecule in a tetrahedral fashion. In addition to the 20 β-­strands comprising the β-helix, there is also a long C-­terminal α-helix. For the first time, Ca²⁺ ions have been observed in addition to the catalytic Zn²⁺ ion. It is hypothesized that Tyr159 (which corresponds to the catalytically important Asn202 in previously reported structures) utilizes C—H...π interactions to fulfill its functions. This study may shed light on the catalytic mechanism of the enzyme and throw open new questions on the mechanism of product removal in carbonic anhydrases.