Cloning, purification, kinetic and anion inhibition studies of a recombinant β-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

Abstract

A β-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAβ has a significant catalytic activity for the physiological reaction, CO₂ + H₂O ⇋ HCO₃⁻ + H⁺ with a k_cat of 1.1 × 10⁵ s⁻¹ and a k_cat/K_m of 7.58 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by acetazolamide (K_I of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAβ at millimolar concentrations, but sulfamide (K_I of 81 µM), N,N-diethyldithiocarbamate (K_I of 67 µM) and sulphamic acid (K_I of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAβ is subsequently proposed as a new drug target for which effective inhibitors can be designed.

Keywords:

• Carbonic anhydrase
• Gyrodactylus salaris
• kinetics
• anion inhibitors
• sulphamic acid

Disclosure statement

CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Data availability statement

Data files pertaining to this study, including the 3 D protein model, are available at https://github.com/MarttiT/G.-salaris-BCA. Individual supplementary files in this repository are indicated as bit.ly links in the text. Further data files and code are stored at https://github.com/thirtysix/Aspatwar.Gsalaris_BCA.

Additional information

Funding

This research was financed by the Italian Ministry for Education and Science (MIUR), grant PRIN: rot. [2017XYBP2R]; Ente Cassa di Risparmio di Firenze (ECRF), grant [CRF2020.1395] (to CTS); Academy of Finland (to SP); Jane & Aatos Erkko Foundation (to SP); Finnish Cultural Foundation (to AA); and Tampere Tuberculosis Foundation (to AA).