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

Figures & data

Figure 1. The acetylcholinesterase inhibitors trichlorfon and dichlorvos.

Figure 2. Schematic presentation of pBVboostFG expression vector designed for production of recombinant protein. The insert contains: 1. attL1, 2. Shine-Dalgarno, 3. Kozak, 4. Met-Ser-Tyr-Tyr, 5. 6 x His, 6. Asp-Tyr-Asp-Ile-Pro-Thr-Thr, 7. Lys-Val, 8. β-CA gene of G. salaris gene of interest, 9. Two stop codons, and 10. AttL2.

Figure 3. Alignment of GsaCAβ sequence with β-CA sequences of other metazoans. The conserved hallmark catalytic-site sequences of β-CAs, CXDXR and HXXC, are shown with black triangles (C: Cysteine, D: Aspartic acid, H: Histidine, R: Arginine, X: any residue). Columns with fully conserved residues are shown as red with white letters. Boxed columns denote positions in which at least 80% of residues are of similar type. The top line shows secondary structures derived from our GsaCAβ model. α: α-helices; β: β-strands; η: 3₁₀-helices; T: turns.

Table 1. Sequences in the multiple sequence alignment of Figure 3.

NCBI accession	Scientific name	Group
KAH8855123.1	Schistosoma japonicum	Platyhelminthes
KAF5400048.1	Paragonimus heterotremus	Platyhelminthes
XP_020895242.1	Exaiptasia diaphana	Sea anemones
GAU97340.1	Ramazzottius varieornatus	Tardigrades
NP_741809.1	Caenorhabditis elegans	Nematodes
XP_037075907.1	Pollicipes pollicipes	Crustaceans
NP_649849.1	Drosophila melanogaster	Insects

Table 2. Percent identity matrix of the aligned protein sequences of Figure 3, as computed by Clustal Omega

		1	2	3	4	5	6	7	8
1	G. salaris	100
2	S. japonicum	30	100
3	P. heterotremus	30	38	100
4	E. diaphana	29	28	29	100
5	R. varieornatus	32	30	29	36	100
6	C. elegans	30	31	33	41	39	100
7	P. pollicipes	31	34	31	44	47	46	100
8	D. melanogaster	28	30	30	41	43	50	60	100

Figure 4. Recombinant GsaCAβ protein analysed on SDS-PAGE. The image shows protein ladder standards (left) and the purified recombinant G. salaris β-CA protein (right) showing a molecular mass calculated from mobility of 26.0 kDa.

Figure 5. Phylogenetic analysis of β-CAs related to the novel enzyme identified in G. salaris.

Table 3. Kinetic parameters for the CO₂ hydration reaction³⁰ catalysed by α- and β-class CA enzymes: the human cytosolic isozymes hCA I and II (α-class CAs) at 20 °C and pH 7.5 in 10 mM HEPES buffer, Can2 (from Cryptococcus neoformans), CalCA (from Candida albicans), SceCA (from Saccharomyces cerevisiae), and Cab (from the archaeon Methanobacterium thermoautotrophicum) measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 10 mM NaClO₄.

Isozyme	Activity level (s^−1)	kcat (M^−1 x s^−1)	kcat/Km (nM)	KI (acetazolamide)
hCA I^a	moderate	2.0 × 10^5	5.0 × 10^7	250
hCA II^a	very high	1.4 × 10^6	1.5 × 10^8	12
Can2^b	moderate	3.9 × 10^5	4.3 × 10^7	10.5
CalCA^c	high	8.0 × 10^5	9.7 × 10^7	132
SceCA ^d	high	9.4 × 10^5	9.8 × 10^7	82
Cab^e	low	3.1 × 10^4	1.82 × 10^6	12100
EcoCAβ^f	moderate	5.3 × 10^5	4.10 × 10^7	227
GsaCAβ^g	low	1.1 × 10^5	7.58 × 10^6	460

^aRef. [10,12,13]; ^bRef.[54,55]; ^cRef. [56–58]; ^dRef. [59,60]; ^eRef.[61,62]; ^fRef. [63–65]; ^gThis work.

Inhibition data with the clinically used sulphonamide, acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulphonamide), are also provided.

Table 4. Anion inhibition data of the β-CA from G. salaris and human isoforms hCA I and hCA II as determined by stopped-flow CO₂ hydrase assay.

	KI (mM)^a
Inhibitor^b	hCA I	hCA II	GsaCAβ
F^−	>300	>300	5.5
Cl^−	6	200	3.3
Br^−	4	63	8.2
I^−	0.3	26	>50
CNO^−	0.0007	0.03	1.9
SCN^−	0.2	1.6	2.7
CN^−	0.0005	0.02	0.86
N3^−	0.0012	1.51	>50
NO2^−	8.4	63	9.1
NO3^−	7	35	>50
HCO3^−−	12	85	>50
CO3^2−−	15	73	>50
HSO3^−−	18	89	6.2
SO4^−	63	>200	>50
HS^−	0.0006	0.04	6.9
NH2SO2NH2	0.31	1.13	0.081
NH2SO3H	0.021	0.39	0.0062
PhAsO3H2	31.7	49	>50
PhB(OH)2	58.6	23	>50
ClO4^−	>200	>200	>50
SnO3^2−	0.57	0.83	0.77
SeO4^2−	118	112	>50
TeO4^2−	0.66	0.92	4.3
OsO5^2−	0.92	0.95	2.5
P2O7^2−	25.8	48	>50
V2O7^2−	0.54	0.57	4.9
B4O7^2−	0.64	0.95	>50
ReO4^−	0.11	0.75	3.8
RuO4^−	0.101	0.69	7.1
S2O8^2−	0.107	0.084	0.91
SeCN^−	0.085	0.086	3.5
NH(SO3)2^2−	0.31	0.76	3.1
FSO3^−	0.79	0.46	0.92
CS3^2−	0.0087	0.0088	5.4
EtNCS2^−	0.00079	0.0031	0.067
PF6^−	>50	>50	>50
CF3SO3^−	>50	>50	>50

^aMean from 3 different assays, by a stopped flow technique (errors were in the range of ± 5–10% of the reported values); ^bSodium salts, except sulfamide and phenylboronic acid.

Figure 6. Molecular model of GsaCAβ. Our model constructed using AlphaFold, yellow, superimposed with pea β-CA (PDB 1EKJ, chain C), sky blue. The zinc ion of the catalytic site is shown in red.

Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem 1971;246:2561–73.

 PubMed Web of Science ®Google Scholar

Supuran CT. Structure and function of carbonic anhydrases. Biochem J 2016;473:2023–32.

 PubMed Web of Science ®Google Scholar

Mishra CB, Tiwari M, Supuran CT. Progress in the development of human carbonic anhydrase inhibitors and their pharmacological applications: where are we today? Med Res Rev 2020;40:2485–565.

 PubMed Web of Science ®Google Scholar

Supuran CT. Exploring the multiple binding modes of inhibitors to carbonic anhydrases for novel drug discovery. Expert Opin Drug Discov 2020;15:671–86.

 PubMed Web of Science ®Google Scholar

Schlicker C, Hall RA, Vullo D, et al. Structure and inhibition of the CO2-sensing carbonic anhydrase Can2 from the pathogenic fungus Cryptococcus neoformans. J Mol Biol 2009;385:1207–20.

 PubMed Web of Science ®Google Scholar

Innocenti A, Muhlschlegel FA, Hall RA, et al. Carbonic anhydrase inhibitors: inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with simple anions. Bioorg Med Chem Lett 2008;18:5066–70.

 PubMed Web of Science ®Google Scholar

Isik S, Guler OO, Kockar F, et al. Saccharomyces cerevisiae β-carbonic anhydrase: inhibition and activation studies. Curr Pharm Des 2010;16:3327–36.

 PubMed Web of Science ®Google Scholar

Isik S, Kockar F, Aydin M, et al. Carbonic anhydrase inhibitors: inhibition of the beta-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates. Bioorg Med Chem 2009;17:1158–63.

 PubMed Web of Science ®Google Scholar

Innocenti A, Zimmerman S, Ferry JG, et al. Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions. Bioorg Med Chem Lett 2004;14:4563–7.

 PubMed Web of Science ®Google Scholar

Zimmerman SA, Ferry JG, Supuran CT. Inhibition of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases. Curr Top Med Chem 2007;7:901–8.

 PubMed Web of Science ®Google Scholar

Data availability statement

Data files pertaining to this study, including the 3 D protein model, are available at https://github.com/MarttiT/G.-salaris-BCA. Individual supplementary files in this repository are indicated as bit.ly links in the text. Further data files and code are stored at https://github.com/thirtysix/Aspatwar.Gsalaris_BCA.