Open Access Highly Accessed Research article

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

Leo Syrjänen123*, Martti Tolvanen1, Mika Hilvo1, Ayodeji Olatubosun1, Alessio Innocenti4, Andrea Scozzafava4, Jenni Leppiniemi1, Barbara Niederhauser1, Vesa P Hytönen1, Thomas A Gorr5, Seppo Parkkila123 and Claudiu T Supuran4

Author Affiliations

1 Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland

2 School of Medicine, University of Tampere and Tampere University Hospital, Tampere, Finland

3 Centre for Laboratory Medicine, Tampere University Hospital, Tampere, Finland

4 Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Sesto Fiorentino (Firenze), Italy

5 Vetsuisse Faculty and Zurich Center for Integrative Human Physiology (ZIHP), Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland

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BMC Biochemistry 2010, 11:28  doi:10.1186/1471-2091-11-28

Published: 26 July 2010



The β-carbonic anhydrase (CA, EC 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 β-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.


The novel β-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of β-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 CO2 hydratase activity, with a kcat of 9.5 × 105 s-1 and a kcat/KM of 1.1 × 108 M-1s-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 (KI values of 0.67 - 1.36 mM) and more potently by sulfamide (KI of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM).


The Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.