Cloning, purification, kinetic and anion inhibition studies of a recombinant β-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

Abstract

A β-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAβ has a significant catalytic activity for the physiological reaction, CO 2 + H 2 O ⇋ HCO 3 − + H + with a k cat of 1.1 × 10 5 s −1 and a k cat /K m of 7.58 × 10 6 M −1 × s −1 . This activity was inhibited by acetazolamide (K I of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAβ at millimolar concentrations, but sulfamide (K I of 81 µM), N,N -diethyldithiocarbamate (K I of 67 µM) and sulphamic acid (K I of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAβ is subsequently proposed as a new drug target for which effective inhibitors can be designed.

WITHDRAWN: Mechanistic modelling of Mycobacterium Tuberculosis beta carbonic anhydrase 3 inhibitors

“…CAs are associated with the performance of various integral physiological functions within living organisms. Firstly, organisms belonging to the taxonomic class of vertebrates are mostly categorized into the α-CA gene family which consists of various molecular conformations of the target enzyme [4,6,[10][11][12][13][14][15][16][17][18][19]. The main biological function of CA includes the reversible hydration of carbon dioxide to bicarbonate and hydrogen ions for maintaining the proper pH homeostasis and their involvement in biosynthetic reactions like gluconeogenesis and ureagenesis in mammals [4].…”

WITHDRAWN: Mechanistic modeling of Mycobacterium tuberculosis β-carbonic anhydrase inhibitors using integrated systems biology and the QSAR approach

“…These proteins, which facilitate the conversion of carbon dioxide to bicarbonate while releasing a proton, play a vital role in various physiological processes related to respiration and the transportation of CO 2 /bicarbonate between metabolizing tissues and lungs [7,8]. They are also involved in maintaining pH and CO 2 balance, regulating electrolyte secretion in different tissues and organs, facilitating biosynthetic reactions (such as gluconeogenesis, lipogenesis, and ureagenesis) [9–19], promoting bone resorption, and influencing tumorigenicity as well as other physiological and pathological processes [7,8,14,19]. Mtb possesses three β-CA genes in its genome: Rv1284 (which encodes a protein referred to as Mtb β-CA1), Rv3588c (which encodes Mtb β-CA2), and Rv3273 (which encodes a third enzyme known as Mtb β-CA3) [19].…”

Sulphonamide inhibition studies of the β-carbonic anhydrase GsaCAβ present in the salmon platyhelminth parasite Gyrodactylus salaris

“…In the last decade, CAs from pathogens started to be considered as possible targets for the development of antiinfectives, for the management of diseases provoked by bacteria 21 , fungi 22 , protozoa 23 and worms 10 , 11 , 24 . In the previous work 1 we have shown that GsaCAβ has a significant catalytic activity for the physiologic, CO 2 hydration reaction, with a k cat of 1.1 × 10 5 s −1 and a k cat / K m of 7.58 × 10 6 M −1 × s −1 . Furthermore, inorganic anions, a well-known class of CA inhibitors (CAIs) 14 , 15 inhibit the enzyme in the millimolar range, as for other α- and β-CAs investigated for their interaction with such modulators of activity 14 .…”

“…We have recently reported the cloning and characterisation of a β-class carbonic anhydrase (CA, EC 4.2.1.1) encoded in the genome of Gyrodactylus salaris , GsaCAβ 1 , a platyhelminth (flatworm) parasite attacking various fish species 2 , 3 . The Atlantic salmon ( Salmo salar ) is particularly sensitive to this parasite, which produced catastrophic losses in fish farms in Scandinavian countries and elsewhere, starting with the 1970s 3–5 .…”

“…Furthermore, inorganic anions, a well-known class of CA inhibitors (CAIs) 14 , 15 inhibit the enzyme in the millimolar range, as for other α- and β-CAs investigated for their interaction with such modulators of activity 14 . Among the investigated such inhibitors, sulfamide (K I of 81 µM) and sulphamic acid ( K I of 6.2 µM) showed the most efficient inhibitory action 1 . Both of them incorporate the SO 2 NH 2 moiety found in the most investigated class of CAIs, the aromatic/heterocyclic sulphonamides and their isosteres (sulfamates, sulfamides) 14 , 15 .…”