Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

Background. The -carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the -CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. Results. The novel -CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of -CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO₂hydratase activity, with a k_catof 9.5 × 10⁵ s^-1 and a k_cat/K_Mof 1.1 × 10⁸ M^{- 1}s^-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (K_Ivalues of 0.67 - 1.36 mM) and more potently by sulfamide (K_Iof 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (K_Is of 26.9 - 43.7 mM). Conclusions. The Drosophila -CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.