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

A new β-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s−1, and kcat/Km of (9.5 ± 0.12) × 107 M−1∙s−1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54–95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.


Introduction
The filamentous ascomycete Sordaria macrospora is a coprophilous fungus that naturally lives on herbivore dung. For many years S. macrospora served as a model organism to study fruiting body development in fungi [1]. Previous studies identified four carbonic anhydrase (CA, EC 4.2.1.1) genes in the genome of S. macrospora that are designated as cas1, cas2, cas3, and cas4 [2][3][4][5]. The two β-CA genes cas1 and cas2 have high sequence identity and encode enzymes with characteristics of the plant-like sub-class of β-CAs [2]. The β-CA cas3 belongs to the cab-like [6] sub-class, whereas cas4 encodes for an α-class CA. CAS1 and CAS3 are cytoplasmic enzymes, while CAS2 is located in the mitochondria, and CAS4 is a secreted protein [3][4][5]. The three β-CAs cas1, cas2, and cas3 are involved in the sexual development of S. macrospora [3]. Deletion of the α-CA cas4 resulted in a significantly reduced rate of ascospore germination but showed no significant involvement in sexual development and vegetative growth [5]. Thus, the detailed physiological roles of all these enzymes are not yet entirely elucidated.

Results and Discussion
The protein encoded by the cas3 gene belongs to the cab-like sub-class of β-CAs and is localized to the cytoplasm [3]. Indeed, the cab-like CAs take their name from the enzyme discovered in the archaeon Methanobacterium thermoautotrophicum, which has only one such enzyme, called cab [9], and they are amongst the smallest CAs ever reported. Similar to cab, CAS3 that is reported here is composed of 174 amino-acid residues, with a calculated molecular mass of 19.2 kDa. CAS3 was synthesized in E. coli Rosetta (DE3) cells as a C-terminal His-tag fusion protein ( Figure 1). After purification, 5-7.5 mg CAS3 could be obtained per L of culture. The purified enzyme was dialyzed against 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 8.3, 50 mM NaCl. To analyze the state of CAS3-His in solution, size-exclusion chromatography and multi-angle laser light scattering (SEC-MALLS) was performed ( Figure 1). The calculated molar mass of CAS3 amounts to 38,650 g·mol −1 (±0.1%), which corresponds to 1.9 times of the CAS3 monomer (20.36 kDa) ( Figure 1). These findings suggest that the biological unit of CAS3 is a homo-dimer in solution, which is the same as for cab and other β-CAs from fungi or bacteria that have been investigated to date [19][20][21][22][23].
The catalytic activity for the physiological CO 2 hydration reaction that is catalyzed by these enzymes was investigated for the purified CAS3 by using a stopped-flow assay [24]. The activity of CAS3 was compared to those of the other two β-CAs present in the genome of this organism (CAS1 and 2), as well as with other similar enzymes from fungi/yeasts (Can2 from Cryptococcus neoformans [22], CalCA from Candida albicans [25], SceCA from Saccharomyces cerevisiae [26]), as well as the widespread human isoforms hCA I and II, belonging to the α-class [7]. As seen from data of Table 1, CAS3 has a catalytic activity that is an order of magnitude higher than CAS1 and CAS2, with the following kinetic parameters: k cat of (7.9 ± 0.2) × 10 5 s −1 , and k cat /K m of (9.5 ± 0.12) × 10 7 M −1 ·s −1 . The activity of CAS3 is, thus, similar to that of CalCA and SceCA, being almost two times higher than that of the "slow" human isoform hCA I, a highly abundant enzyme in red blood cells [27]. Only hCA II among the investigated enzymes shown in Table 1 has a higher catalytic activity, but this enzyme is known to be one of the most effective catalysts in nature [7][8][9]. In addition, the CO 2 hydrase activity was effectively inhibited by the clinically used sulfonamide acetazolamide, one of the most investigated CA inhibitors (CAIs) to date [7,8] (Table 1). Thus, apparently, CAS3 also has a higher affinity for sulfonamide inhibitors compared to CAS1 and 2, for which acetazolamide was a rather weak inhibitor, with K I s in the range of 445-816 nM (Table 1) [17,18]. that of the "slow" human isoform hCA I, a highly abundant enzyme in red blood cells [27]. Only hCA II among the investigated enzymes shown in Table 1 has a higher catalytic activity, but this enzyme is known to be one of the most effective catalysts in nature [7][8][9]. In addition, the CO2 hydrase activity was effectively inhibited by the clinically used sulfonamide acetazolamide, one of the most investigated CA inhibitors (CAIs) to date [7,8] (Table 1). Thus, apparently, CAS3 also has a higher affinity for sulfonamide inhibitors compared to CAS1 and 2, for which acetazolamide was a rather weak inhibitor, with KIs in the range of 445-816 nM (Table 1) [17,18]. The calculated molar mass of CAS3 amounts to 38,650 g•mol −1 (±0.1%), which corresponds to 1.9 times of the CAS3 monomer (20.36 kDa) (C). Peak 1 in the CAS3 elution profile is very likely due to protein aggregates that eluted close to the void volume of the column. Peak 2 represents the CAS3-His protein. Table 1. Kinetic parameters for the CO2 hydration reaction [24] catalyzed by the human cytosolic enzymes hCA I and II (α-class CAs) at 20 °C and pH 7.5, and the β-CAs Can2, CalCA (from Cryptococcus neoformans and Candida albicans, respectively), SceCA (from Saccharomyces cerevisiae), as well as the enzymes from Sordaria macrospora, CAS1-CAS3, measured at 20 °C, pH 8.3. Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) are also provided.
As seen from data of     Figure 2. Structure of sulfonamide and sulfamate inhibitors of types 1-24 and AAZ-HCT investigated in the present study. Table 2. Inhibition of human isoforms hCA I and hCA II, and of the β-class fungal enzymes CAS1-CAS3 with sulfonamides 1-24 and the clinically used drugs AAZ-HCT, by a stopped flow CO 2 hydrase assay [24].  [7,8]; b from reference [17]; c this work: mean ± standard error, from three different assays.

Construction of the cas3 Overexpression Vector
RNA from S. macrospora was isolated as described in Reference [3]. To remove obsolete genomic DNA, the RNA was treated with DNase I (Thermo Scientific, EN0521, Waltham, MA, USA) according to the manufacturer's manual. The "Transcriptor High Fidelity cDNA Synthesis kit" (Roche, Basil, Switzerland) was used for the reverse transcription reaction. Template concentration was 2 µg of RNA. The complementary DNA (cDNA) of the cas3 open reading frame (ORF; 525 bp) was amplified with primer pair CAS-pet-f (GGAGATATACATAATGATGCCCGTCACCAACGAAGA) and Cas3-pet-r (GGTGGTGGTGCTCGAGAACAACCCTCACCGTCTTGC). The obtained PCR fragment was cloned into the pET22b(+) expression plasmid (Novagen, Madison, WI, USA) linearized with NdeI and XhoI to create a 6×His fusion protein. The plasmid was named pET-CAS3.

Heterologous Expression of the cas3 Gene in E. coli
The production of the CAS3 protein was performed in E. coli strain Rosetta (DE3) (Invitrogen, Carlsbad, Germany). An overnight pre-culture was used to inoculate 4 × 0.5 L of Luria broth (LB) medium supplemented with 100 mg/L ampicillin and 0.5 mM ZnSO 4 . The cultures were grown to an OD 600 of 0.1. Heterologous gene expression was then induced by the addition of 1 mM IPTG during the exponential phase of growth and lasted for 3-4 h at 30 • C. Subsequently, the cells were harvested (4000× g, 30 min, 4 • C) and flash-frozen with liquid nitrogen and stored at −20 • C.
For determination of the protein concentration, 10 µL of the protein was mixed with 990 µL of Bradford reagent [35] and incubated for 2 min at RT. Then, the absorption was determined at 595 nm in a "Libra S12" (biochrom, Cambridge, UK) spectrophotometer in a 1-mL cuvette. Prior to this, a calibration line with bovine serum albumin was recorded.

Size-Exclusion Chromatography Coupled with Multi-Angle Laser Light Scattering
Four hundred microliters CAS3 (3 mg/mL) were loaded separately on a 24-mL analytical "Superdex 200 (10/300)" gel filtration column using an "Äkta purifier" coupled to a "miniDAWN MALS" detector (Wyatt Technology, Santa Barbara, CA, USA). The column was pre-equilibrated with 50 mM NaH 2 PO 4 pH 8.0, 250 mM imidazole, 300 mM NaCl. Multi-angle laser light-scattering analysis was performed continuously on the column eluate at 291 K (size-exclusion chromatography coupled with multi-angle laser light scattering, SEC-MALLS). Data analysis was carried out with Astra software (Wyatt Technology, Santa Barbara, CA, USA).

CA Inhibition Assay
An Applied Photophysics stopped-flow instrument was used for assaying the CA catalyzed CO 2 hydration activity [24]. The pH indicator used was bromothymol blue at a concentration of 0.2 mM, working at the absorbance maximum of 557 nm. The buffer used was 20 mM Tris (pH 8.3), and 20 mM Na 2 SO 4 was added to this buffer for maintaining constant the ionic strength (sulfate is not inhibitory and has a K I > 200 mM against this enzyme; data not shown). The initial rates of the CA-catalyzed CO 2 hydration were followed for a period of 10-100 s for each assay. The CO 2 concentrations ranged from 1.7 to 17 mM for determining kinetic parameters of the reaction and the inhibition constants of tested inhibitors. For each measurement, six traces of the initial 5%-10% of the reaction were used for determining the initial velocity. Then, 10-fold decreasing dilution of inhibitor solutions, ranging from 1 nM and 100 µM were employed for determining the inhibition constants. Uncatalyzed rates were determined in the same manner and subtracted from total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled deionized water, and dilutions up to 1 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature before to assay for allowing for the formation of E-I complexes. The inhibition constants were obtained by non-linear least-squares methods using the Cheng-Prusoff equation, and they represent the mean from at least three different determinations. The human isoforms hCA I, II were assayed in the same conditions as above except that the working pH was 7.4 HEPES buffer with phenol red as an indicator [21,22]. The inhibition constants measured by this method are in excellent agreement with the dissociation constants measured by native mass spectrometry [37,38].

Conclusions
Fungal CAs are of great interest both for biotechnological and pharmaceutical applications [10] because some of these enzymes are stable, relatively easy to produce, and may be used as model enzymes for testing inhibitors/activators. The organism Sordaria macrospora, used as a genetic model to study fruiting body development of filamentous fungi, encodes for at least four different CAs, two of which were thoroughly investigated earlier, CAS1 and CAS2. Here, we prove that CAS3, another representative enzyme from this organism, belonging to the β-CA class, may be of interest for better understanding the roles these proteins play in various physiologic processes of fungi. Unlike CAS1 and CAS2, which showed rather low catalytic activity for the hydration of CO 2 to bicarbonate and protons, CAS3 is a highly effective catalyst, showing kinetic parameters comparable to those of other fungal/mammalian enzymes, i.e., k cat of (7.9 ± 0.2) × 10 5 s −1 , and k cat /K m of (9.5 ± 0.12) × 10 7 M −1 ·s −1 . A detailed inhibition study of CAS3 with sulfonamides and one sulfamate allowed us to demonstrate that this enzyme has higher affinity for these inhibitors compared to CAS1 and CAS2, with clinically used agents such as benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfamilamide, metanilamide, and benzene-1,3-disulfonamide possessing K I s under 100 nM.