Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates
Innocenti, Alessio; Scozzafava, Andrea; Gorr, Thomas A; Syrjänen, Leo (Tay); Tolvanen, Martti (Tay); Hilvo, Mika (Tay); Olatubosun, Ayodeji (Tay); Leppiniemi, Jenni (Tay); Niederhauser, Barbara (Tay); Hytönen, Vesa P (Tay); Parkkila, Seppo (Tay) (2010)
2010
BMC Biochemistry 11
28
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
Julkaisun pysyvä osoite on
https://urn.fi/urn:nbn:uta-3-513
https://urn.fi/urn:nbn:uta-3-513
Kuvaus
BioMed Central Open access
Tiivistelmä
Background
The β-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 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.
Results
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).
Conclusions
The Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.
The β-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 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.
Results
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).
Conclusions
The Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.
Kokoelmat
- Artikkelit [6140]