Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-36011
Syrjänen, L; Tolvanen, M; Hilvo, M; Olatubosun, A; Innocenti, A; Scozzafava, A; Leppiniemi, J; Niederhauser, B; Hytönen, V P; Gorr, T A; Parkkila, S; Supuran, C T (2010). Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates. BMC Biochemistry, 11:28.
BACKGROUND: The beta-carbonic anhydrase (CA, EC 220.127.116.11) 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 beta-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 beta-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of beta-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 x 105 s-1 and a kcat/KM of 1.1 x 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). CONCLUSIONS: The Drosophila beta-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.
|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||05 Vetsuisse Faculty > Institute of Veterinary Physiology|
|DDC:||570 Life sciences; biology|
|Deposited On:||11 Nov 2010 12:38|
|Last Modified:||19 Jul 2014 13:47|
|Citations:||Web of Science®. Times Cited: 20|
Scopus®. Citation Count: 21
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