Zeta-carbonic anhydrases show CS2 hydrolase activity: A new metabolic carbon acquisition pathway in diatoms?

Abstract

CDCA1 is a very peculiar member of the Carbonic Anhydrase (CA) family. It has been the first enzyme to show an efficient utilization of Cd(II) ions in Nature and a unique adaptation capability to live on the surface ocean. Indeed, in this environment, which is extremely depleted in essential metal ions, CDCA1 can utilize Zn(II) or Cd(II) as catalytic metal to support the metabolic needs of fast growing diatoms. In this paper we demonstrate a further catalytic versatility of this enzyme by using a combination of X-ray crystallography, molecular dynamics simulations and enzymatic experiments. First we identified the CO₂ binding site and the way in which this substrate travels from the environment to the enzyme active site. Then, starting from the observation of a structural similarity with the substrate entry route of CS₂ hydrolase from Acidanius A1-3, we hypothesized and demonstrated that also CS₂ is a substrate for CDCA1. This finding is new and unexpected since until now only few CS₂ hydrolases have been characterized, and none of them is reported to have any CO₂ hydratase action. The physiological implications of this supplementary catalytic activity still remain to be unveiled. We suggest here that it could represent another ability of diatoms expressing CDCA1 to adapt to the external environment. Indeed, the ability of this enzyme to convert CS₂ could represent an alternative source of carbon acquisition for diatoms, in addition to CO₂.

Keywords: AAZ, Acetazolamide; CA, Carbonic Anhydrase; CAI, Carbonic Anhydrase Inhibitor; CCD, Charge Coupled Device; CDCA1, Cadmium-specific Carbonic Anhydrase; CO2; CS2; CS2H, S. solfataricus CS2 hydrolase; Cambialistic enzyme; Carbonic Anhydrase; DMSO, Dimethyl Sulfoxide; FbiCA, Flaveria bidentis Carbonic Anhydrase; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; IPTG, Isopropyl-β-D-1-thiogalactopyranoside; MD, Molecular Dynamics; Molecular dynamics; NCS, Non-Crystallographic Symmetry; PDB, Protein Data Bank; PEG, Polyethylene glycol; SDS-PAGE, Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis; Tris-HCl, Tris(hydroxymethyl)aminomethane hydrochloride; bCA, bovine Carbonic Anhydrase; hCA, human Carbonic Anhydrase; psCA3, Pseudomonas aeruginosa Carbonic Anhydrase 3.