Cloning, Characterization and Anion Inhibition Studies of a β-Carbonic Anhydrase from the Pathogenic Protozoan Entamoeba histolytica

Abstract

We report the cloning and catalytic activity of a β-carbonic anhydrase (CA, EC 4.2.1.1), isolated from the pathogenic protozoan Entamoeba histolytica, EhiCA. This enzyme has a high catalytic activity for the physiologic CO2 hydration reaction, with a kcat of 6.7 × 105 s−1 and a kcat/Km of 8.9 × 107 M−1 × s−1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules revealed that fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (KIs of 0.51–8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (KIs in the range of 28–86 μM). Since β-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis

“…These data, together with the observation that b-CAs are not present in humans, indicate the latter enzymes as excellent targets for the development of new antiparasitic drugs. However, despite their growing importance, only a few papers on the kinetics and inhibition profiles of b-CAs have been published [62][63][64] , and no crystal structures of a protozoan CA have so far been reported. Here, we illustrate a full biochemical characterisation of TvaCA1, together with its crystallographic structure, thus providing the first detailed characterisation of a protozoan b-CA.…”

Anion Inhibition Studies of the β-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora

“…Trithiocarbonate has a K I s in the range of 8.7-9.9 µM for human (h) isoforms hCA I-III, 36.15 mM for hCA VII and 0.43 mM for hCA XIII [6], whereas the three classes of organic inhibitors achieved low nanomolar inhibition for pharmaceutically relevant CA isoforms, such as hCA I, II, IV, IX and XII [9][10][11][12][13]. Thus, many anion inhibitors have been investigated for their interaction with CAs from various organisms, such as mammals (humans) [4,7,8,14], bacteria [15][16][17][18], protozoans [19][20][21], corals [22] fungi [23][24][25][26][27] and Archaea [28]. Fungi encode for CAs belonging to the αand β-CA genetic families (of the eight CA classes presently known [29]) and these enzymes play crucial roles in the growth, development, virulence and survival of these organisms [30,31].…”

Activation Studies of the γ-Carbonic Anhydrases from the Antarctic Marine Bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea with Amino Acids and Amines

“…Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloproteins involved in several metabolic processes [8,9,10,11,12,13,14,15,16,17] either directly, by providing CO 2 /bicarbonate for carboxylating reactions [8,9,12,13], or indirectly, by modulating pH [13,14,15,16,17]. Indeed, CAs convert the neutral molecules CO 2 and water to a weak base (bicarbonate) and a strong acid (hydronium ion) with very high efficacy [8,13].…”

“…For this reason, in many organisms including bacteria, CAs are the main players in pH homeostasis and related physiologic processes, which are involved in the metabolism, survival and colonization of various niches in which these organisms thrive [18,19,20,21,22,23,24,25]. These processes were mainly investigated for pathogenic microorganisms, such as bacteria [9,13,16,17], protozoa [10,11,21,22] and fungi [17,26]. However, the wide distributions of these enzymes in non-pathogenic Archaea [25] and Bacteria [3,4,6,7,27] make them of interest for understanding crucial biochemical processes connected with metabolism, adaptation to various environmental conditions, and survival in extreme environments.…”