Functional characterization of CAS1, CAS2, CAS3 and CAS4

3.5.1 Complementation of a CA-deficient S. cerevisiae ∆nce103 deletion mutant

The budding yeast S. cerevisiae contains only the single cytoplasmic plant-like β-CA NCE103p.

The respective haploid deletion mutant ∆nce103 exhibits a HCR phenotype and cannot grow in ambient air. Heterologous expression of CA genes can rescue the S. cerevisiae ∆nce103 deletion mutant (Götz et al. 1999), with complementation depending on carbonic anhydrase activity of the heterologous enzyme (Clark et al. 2004). The cDNA of all four cas genes was expressed in the yeast mutant. The native CAS2 protein exhibits an N-terminal signal peptide (MTS) for translocation into mitochondria (Elleuche and Pöggeler 2009b). To enable cytoplasmic localization of cas2 in S. cerevisiae, the nucleotides of the cas2 cDNA encoding the MTS were removed. The CAS4 protein exhibits an N-terminal signal sequence (SS) for translocation into the endoplasmic reticulum that was removed for expression in S. cerevisiae. To demonstrate the ability of CAS1, CAS3, and of the truncated CAS2 and CAS4 to functionally restore the CA-deficient yeast strain, the corresponding four genes were expressed in the haploid deletion strain CEN.HE28-h (∆nce103) (Table 1) (Fig. 26).

Fig. 26: Functional complementation of the haploid S. cerevisiae CA deletion mutant ∆nce103 with cas1, cas2, cas3 and cas4 of S. macrospora. The haploid yeast deletion strain CEN.HE28-h (∆nce103) is only able to grow under high CO2 levels. It was transformed with galactose-inducible plasmids p426-CAS1-His, p426-CAS2-His, p426-CAS3-His and p426-CAS4-p426-CAS3-His. Recombinant strains were grown three days at 30 °C in a 5% CO2 enriched atmosphere as viability control and at 30 °C in ambient air on SD-Ura and SG-Ura plates for repression and induction of gene expression. As controls the haploid as well as the heterozygous diploid strain (CEN.HE28) were transformed with the empty vector p426GAL1. Complementation was confirmed when growth occurs in ambient air on SG-Ura plates.

Western blotting with an anti-His antibody was performed to confirm the production of the heterologous proteins.

SG-Ura

Results 78 Only the full length cDNA of cas1 and a truncated version of the cas2 cDNA lacking the mitochondrial target sequence (MTS) fully complemented the phenotype of the S. cerevisiae

∆nce103 CA mutant, demonstrating the carbonic anhydrase activity of the S. macrospora β-CAs CAS1 and CAS2 (Fig. 26). Neither the cab-like β-CA CAS3 nor the α-class CA CAS4 could restore the CA-deficient yeast strain (Fig. 26). The haploid deletion strain transformed with the empty vector served as negative control while the heterozygous strain (CEN.HE28) transformed with the empty vector was used as positive control. In addition, all strains were tested for their viability by incubation on SD-Ura at 5% CO2 (Fig. 26). To verify the production of both proteins, western blotting with anti-His antibody was performed (Fig. 26).

The results are in agreement with the expected fragment sizes for all four proteins (CAS1:

26.2 kDa; CAS2: 26.9kDa; CAS3: 20.3 kDa and CAS4: 38.4 kDa)

3.5.2 Analysis of the in-vitro activity and inhibition of CAS1 and CAS2

CAS1 is a cytosolic protein composed of 234 aa with a calculated molecular weight of 25.1 kDa. The native CAS2 protein (284 aa) exhibits an N-terminal signal peptide for translocation into mitochondria (Elleuche and Pöggeler 2009b). The MTS is predicted to be cleaved between His59 and Ser60 (Elleuche and Pöggeler 2009a). Therefore, the nucleotides encoding the MTS were removed to enable expression of cas2 in E. coli. The truncated cas2 gene expressed in E. coli encodes a protein of 225 residues with a calculated molecular weight of 25.9 kDa. CAS3 localizes to the cytoplasm and is composed of 174 aa with a calculated molecular mass of 19.2 kDa. The CAS4 protein (368 aa) exhibits an N-terminal signal sequence for translocation into the endoplasmic reticulum. The SS is predicted to be cleaved between Ser21 and Leu22. For expression in E. coli the sequence encoding the SS was removed. The mature, secreted CAS4 protein consists of 347 aa with a predicted molecular mass of 37.2 kDa. CAS1, CAS2, CAS3 and CAS4 were synthesized in E. coli Rosetta (DE3) cells as N- or C-terminal His-tag fusions. While CAS1 was N-terminally His-tagged, CAS2, CAS3 and CAS4 had been C-terminally His-tagged. The overexpression of all four cas genes was successful and beside of CAS4 all CAS proteins could also be purified (Fig. 27). After purification, 5-10 mg of CAS1, 10-20 mg of CAS2 and 5-7.5 mg CAS3 could be obtained per L of culture. The purified enzymes CAS1 and CAS2 were concentrated to 10 mg mL⁻¹ and dialyzed against 50 mM HEPES pH 8.3, 50 mM NaCl, and tested in a stopped-flow CO₂ hydration assay as described in 2.2.5.6.

Fig. 27: Purification of His-CAS1 (A), CAS2-His (B) and CAS3-His (C). Coomassie stained, 15% SDS-Gel of purified His-CAS1, CAS2-His and CAS3-His. After washing of unbound proteins the His-tagged enzymes were eluted by addition of 500 µL elution buffer. 10 µL of the protein solution were separated in a SDS-PAGE.

Expected fragment sizes are 27.34 kDa (CAS1), 26.91 kDa (CAS2) and 20.36 kDa (CAS3).

CAS1 and CAS2 exhibit measurable in-vitro CO₂ hydrase activity (k_cat/K_m of CAS1: 1.30 × 10⁶ M⁻¹ s⁻¹; CAS2: 1.21 × 10⁶ M⁻¹ s⁻¹; Table 6).

In addition, CAS1 and CAS2 were only weakly inhibited by the widely used sulfonamide drug acetazolamide, with inhibition constants of 445 nM and 816 nM against CAS1 and CAS2, respectively (Table 6).

Table 6: Overview about the kinetic parameters of different α- and β-class carbonic anhydrases derived from a CO2 hydration assay (Khalifah 1971).

Isozyme Activity level k_cat (s⁻¹) k_cat/K_m (M⁻¹ s⁻¹) K_I (acetazolamide) (nM) S. cerevisiae), FbiCA 1 (from the plant Flaveria bidentis) and CAS1 and CAS2 of S. macrospora measured at 20

°C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4. Inhibition data with the clinically used sulfonamide

Results 80 Inhibition by anions was also investigated, as these have been shown to effectively inhibit CA activity (De Simone and Supuran 2012). The majority of the anions tested were ineffective at inhibiting CAS1 and CAS2 (Table S2).

Perchlorate and tetrafluoroborate showed weak inhibition, similarly to several other CAs (Vullo et al. 2013b). Nitrite and nitrate anions were also ineffective CAS1 and CAS2 inhibitors with inhibition constants over 100 mM. The halogens bromide and chloride inhibited CAS1 with inhibition constants of 9.3 and 9.2 mM, respectively, while CAS2 was much more weakly inhibited. Conversely, CAS2 was more strongly inhibited by sulfate (KI = 4.8 mM) than was CAS1 (KI > 100 mM). The best anionic inhibitors were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with inhibition constants from 84 to 9 µM for CAS1 and from 72 to 48 µM for CAS2 (Table S2).