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 beta-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 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.
Syrjänen
Leo
L
Institute of Medical Technology, University of Tampere and Tampere University Hospital, Finland. leo.syrjanen@uta.fi
Tolvanen
Martti
M
Hilvo
Mika
M
Olatubosun
Ayodeji
A
Innocenti
Alessio
A
Scozzafava
Andrea
A
Leppiniemi
Jenni
J
Niederhauser
Barbara
B
Hytönen
Vesa P
VP
Gorr
Thomas A
TA
Parkkila
Seppo
S
Supuran
Claudiu T
CT
D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D030562:Databases, Protein; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007697:Kinesics; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology
D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013449:Sulfonamides; D002256:Carbonic Anhydrases