Escherichia coli γ-carbonic anhydrase: characterisation and effects of simple aromatic/heterocyclic sulphonamide inhibitors

Abstract Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes involved in biosynthetic processes, transport, supply, and balance of CO2/HCO3- into the cell. In Bacteria, CAs avoid the depletion of the dissolved CO2/HCO3- from the cell, providing them to the central metabolism that is compromised without the CA activity. The involvement of CAs in the survival, pathogenicity, and virulence of several bacterial pathogenic species is recent. Here, we report the kinetic properties of the recombinant γ-CA (EcoCAγ) encoded in the genome of Escherichia coli. EcoCAγ is an excellent catalyst for the physiological CO2 hydration reaction to bicarbonate and protons, with a kcat of 5.7 × 105 s−1 and kcat/KM of 6.9 × 106 M−1 s−1. The EcoCAγ inhibition profile with a broad series of known CA inhibitors, the substituted benzene-sulphonamides, and clinically licenced drugs was explored. Benzolamide showed a KI lower than 100 nM. Our study reinforces the hypothesis that the synthesis of new drugs capable of interfering selectively with the bacterial CA activity, avoiding the inhibition of the human α -CAs, is achievable and may lead to novel antibacterials.


Introduction
Nature developed a fascinating system for recycling CO 2 1,2 . Plant, algae, and photosynthetic prokaryotes through the photosynthetic process can convert light energy into chemical energy 1 . The last is stored in the carbohydrates, and the carbon dioxide (CO 2 ) is fixed into biomass. In the aerobic environment, carbon from the biomass returns to the atmosphere by the action of O 2 -requiring decomposers, such as bacteria and fungi 3 , while, in an environment characterised by the absence of oxygen, the anaerobic microbes decompose the biomass releasing methane and CO 2 4 .
The carbon cycle is essential for the life on the Earth since carbon is a critical component in controlling the planet temperature, a crucial food ingredient for sustaining the entire living organisms, and, finally, an energy source for driving the global economy 5 . A necessary enzyme involved in the carbon cycle, which has the function to enhance the photosynthesis via a mechanism that concentrates and supplies CO 2 up to 1000-fold close to the ribulose-1,5-bisphosphate carboxylase (RuBisCO), is the carbonic anhydrase (CA, EC 4.2.1.1) 6 . Several CA-classes have been identified, such as a, b, c, d, f, g, h, and i [7][8][9] . The eight CA-classes, showing a low sequence similarity, different folds and structures, are considered phylogenetically distinct. This exceptional sequence and structural divergence evolved, reflecting a convergent evolution of the CA-classes since they target a common substrate, the CO 2, and catalyse the same reaction, the simple but physiologically crucial reaction of carbon dioxide hydration/dehydration to bicarbonate and protons (CO 2 þ H 2 O À HCO 3 þ H þ ) [10][11][12][13][14][15][16][17] . The CO 2 hydratase reaction is catalysed at very high rates, with a pseudo-first-order kinetic constant (k cat ) ranging from 10 4 to 10 6 s À1 (18,19), which is about 67,000-7,000,000 times higher than the uncatalyzed reaction with a k cat ¼0.15 s À118, 19. In addition to the involvement in photosynthetic processes, CAs accomplish the transport, supply, and balance of CO 2 and bicarbonate into the cell, but also pH homeostasis, secretion of electrolytes, and participate in several biosynthetic processes 20,21 . The homeostasis of H þ and CO 2 /HCO 3 is involved in many physiological and pathological conditions 18,19,[22][23][24][25][26] . In Bacteria, for example, the primary CA function is to avoid the depletion of the dissolved inorganic carbon (CO 2 /HCO 3 -) from the cell, providing them quickly to the central metabolism, which might be compromised without the CA activity 7,8,16,[27][28][29] . A charming example is represented by the b-CA (CynT) encoded by the genome of the bacterium Escherichia coli, a Gram-negative bacterium typically colonising the lower intestine of warm-blooded organisms [30][31][32] . The E. coli cyn operon contains three genes, CynT, CynS, and CynX encoding for a b-CA, a cyanase, and an unknown protein, respectively 33 . CynT catalysing the CO 2 hydration produces HCO 3 -, whose depletion from the bacterial cells is avoided since the cyanase uses it as a substrate to produce ammonia and CO 2 33-35 . Numerous examples support the importance of CA activity in the growth of bacteria. For example, the deletion of a gene encoding for the b-CA from Ralstonia eutropha allowed the heterotrophic growth of the mutant only at an elevated concentration of CO 2 compared to wild-type 36 . In E. coli, a second b-CA, CynT2, is essential for the growth of the microorganism at atmospheric pCO 2 such as those belonging to the genera Buchnera and Rickettsia, determined their adaptation only in high-CO 2 niches 39 . More interesting, are the discovery of the involvement of CAs in the survival, pathogenicity, and virulence of several species of human pathogens, such as Helicobacter pylori [40][41][42] , Vibrio cholerae 43 , Brucella suis [44][45][46][47] , Salmonella enterica 48 , and Pseudomonas aeruginosa 49 among others.
In this context, we focalised our interest in the inhibition profile of the CAs encoded by the genome of E. coli, a bacteria generally coexisting in a mutually beneficial state with the host 50 . In some cases, it may become a severe pathogen [51][52][53] or can cause diseases if the host defences are weakened 54 . The E. coli genome encodes for b-CAs (Cyn T and CynT2), cand i-CAs. Here, we cloned, overexpressed, and purified the c-CA (EcoCAc) enzyme. The recombinant EcoCAc was subjected to the investigation of its kinetic properties since only the three-dimensional structure was determined in 2012 55 . Besides, we explored the EcoCAc inhibition profile with a broad series of substituted benzene-sulphonamides and clinically licenced drugs, which generally inhibit the CAs in the nanomolar range [56][57][58] . The EcoCAc inhibition profile was compared with those obtained for the two human isoforms (hCA I and hCA II) with the prospect of gaining new scientific knowledge in the design of potentially new inhibitors capable of blocking efficiently and selectively the CA activity encoded by the pathogenic microbes.

Chemicals and instruments
All the chemicals used in this study were of reagent grade and purchased from Sigma. The Affinity column (His-Trap FF) and the AKTA-Prime purification system were bought from GE Healthcare. The SX20 Stopped-Flow and SDS-PAGE and Western-Blot apparatus were obtained by AppliedPhotophysics and BioRAD, respectively.

Cloning, expression and purification of the recombinant E.coli c-CA
The synthetic E. coli gene encoding for the EcoCAc (Accession number: WP_009008373.1) was synthesised by the Invitrogen GeneArt (ThermoFisher Scientific), a company specialised in gene synthesis, and cloned into the expression vector pET100D-Topo/ c-CA. Briefly, the gene was designed to produce the recombinant EcoCAc as fusion proteins with a tag containing nucleotides encoding for six histidines (His-Tag) at the amino terminus of neosynthetized recombinant protein. Competent E. coli BL21 (DE3) codon plus cells (Agilent) were transformed as described by Del Prete et al. 59 . Isopropyl b-D-1-thiogalactopyranoside (IPTG) at the concentration of 1 mM was added to the cellular culture to overexpress the recombinant EcoCAc. After growth, the cells were harvested and disrupted by sonication. Cellular extract was purified using a nickel affinity column (His-Trap FF), which allows the interaction between the matrix functionalised with Ni 2þ ions and the His-Tag at the N-terminus of the protein. The HisTrap column (1 ml) was equilibrated with 20 ml equilibration buffer (50 mM Tris, 20 mM imidazole and 150 mM sodium chloride, pH 7.5) at 1 ml/ min. The supernatant from the cellular lysate was loaded onto the column at 1 ml/min, and eluted from the column by fluxing imidazole (300 mM) at a flow of 0.5 ml/min in a buffer composed of 50 mM Tris and 300 mM sodium chloride, pH 7.5. The recovered EcoCAc was 90% pure. The protein quantification was carried out by Bradford method (BioRAD) 60 .

Enzyme activity for monitoring the EcoCAc purification
The CA activity assay was performed as described by Capasso et al. 61 . Briefly, the assay was based on the monitoring of pH variation due to the catalysed conversion of CO 2 to bicarbonate. Bromothymol blue was used as the indicator of pH variation. The assay was performed at 0 C and a CO 2 -satured solution was used as substrate. The enzyme activity was calculated by measuring the time required for Bromothymol blue to change from blue to yellow. This time is inversely related to the quantity of enzyme present in the sample and allows the calculation of the Wilbur-Anderson units as described previously 61 .

Sds-PAGE, protonography and Western-Blot
A 12% Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prepared as described by Laemmli 62 was used, loading on the gel the recovered EcoCAc from the affinity column. The gel was stained with Coomassie Brilliant Blue-R. To perform the protonography, wells of 12% SDS-PAGE gel were loaded with samples mixed with loading buffer not containing 2-mercaptoethanol and not subjected to boiling, in order to avoid protein denaturation. The gel was run at 150 V until the dye front ran off the gel. Following the electrophoresis, the 12% SDS-PAGE gel was subject to protonography to detect the yellows bands due to the hydratase activity on the gel as described previously [63][64][65][66] . In addition, EcoCAc was transferred to a PVDF (polyvinylidene fluoride) membrane with transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) using Trans-Plot SD Cell (Bio-Rad, Hercules, CA, USA). His-Tag Western blot was carried out using the Pierce Fast Western Blot Kit (Thermo Scientific, Waltham, MA, USA). Blotted membrane was placed in the wash blot solution Fast Western 1 Wash Buffer to remove transfer buffer. Primary Antibody Working Dilution was added to the blot and incubated for 30 min at room temperature (RT) with shaking. Invitrogen anti-His antibody (1:10000) was used. Afterwards, the blot was removed from the primary antibody solution and incubated for 10 min with the FastWestern Optimised HRP ReagentWorking Dilution. Subsequently, the membrane was washed two times in about 20 ml of FastWestern 1Wash Buffer. Finally, the membrane was incubated with the detection reagent working solution and incubated for 1 min, at room temperature, and then developed with X-ray film.

Kinetic parameters and inhibition constants
The CO 2 hydration activity performed by the EcoCAc was monitored using an Applied Photophysics stopped-flow instrument 67 . Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 20 mM TRIS (pH 8.3) as buffer, and 20 mM NaClO 4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO 2 hydration reaction for a period of 10-100 s. To determine the kinetic parameters by Lineweaver-Burk plots and the inhibition constants, a concentration of CO 2 between 1.7 to 17 mM was used. At least six measurements of the original 5-10% reaction were used to assess the initial velocity for each inhibitor. The uncatalyzed rates were identically determined and detracted from the total observed rates. Stock inhibitor solutions (10-100 mM) were prepared in distilled-deionized water and dilutions up to 0.01 mM were done with the buffer test. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex or for the eventual active site mediated hydrolysis of the inhibitor. The inhibition constants were obtained by non-linear least-squares methods using PRISM 6 and the Cheng-Prusoff equation, as reported earlier 12,14,68 , and represent the mean from at least three different determinations. h CAI and hCA II were recombinant enzymes obtained in-house.

Primary structure analysis
Because the genome of Escherichia coli was full sequenced, it was designed and polymerised the synthetic gene encoding for the EcoCAc polypeptide chain. The sequence of the synthetic gene and its corresponding protein are shown in Figure 1. The EcoCAc nucleotide sequence consists of an open reading frame of 771 nucleotides, encoding for a polypeptide chain of 256 amino acid residues ( Figure 1). The EcoCAc was aligned with the YrdA amino acid sequence, which corresponded to the c-CA crystallised in 2012 (55). As shown in Figure 2, EcoCAc has the same polypeptide chain of the YrdA protein, except for the presence of 72 additional amino acids at the N-terminus. In bacteria genes are often found in a cluster on the chromosome, which are under control of a single promoter 69 . This gene organisation is known as an operon. Thus, it is possible that the additional 72 residues belong to a different protein encoded by the E. coli operon, which includes the gene encoding for the E.coli c-CA, too. As described in the literature, c-CA is a homotrimer with three zinc-containing active sites located at the interfaces between two monomers 55 . Each monomer is structurally characterised by a tandemly-repeated hexapeptide, which mostly shows an aliphatic residue, usually Ile, Val, or Leu, at the first position, a well-conserved residue is glycine at position two, and a residue Ala, Ser, Cys, Val, Thr, or Asn at position five (Figure 2) 70 . This repeated hexapeptide is essential for the left-hand fold of the trimeric b-helix structures, which contradistinguish the canonical c-CAs and putative acetyltransferases/ acyltransferases 55 . YrdA was crystallised without the extra 72 residues at the N-terminus because the authors considered only the polypeptide chain with the conserved hexapeptide-repeat motifs (182 amino acid residues), which generally typify all the c-CA sequences ( Figure 2).
Next paragraphs report 1) the heterologous expression and purification of the EcoCAc full amino acid sequence (containing the extra 72 residues at the N-terminus of the polypeptide chain); 2) the determination of the kinetic parameters of EcoCAc using the stopped-flow technique; and 3) the inhibition profile of EcoCAc with a broad range of small molecules, which generally inactivate this class of enzyme.   containing the complete EcoCAc gene resulted in the production of the recombinant c-CA as a chimeric polypeptide chain obtained by the fusion of the N-terminus of the EcoCAc protein to the Cterminus of a soluble peptide (about 4.0 kDa) containing six histidines (His-Tag). This strategy has been adopted to improve the solubility, purification, and detection of the recombinant EcoCAc expressed in its complete form (plus the extra 72 residues at the N-terminus). After the sonication and centrifugation, most of the CA activity was recovered in the soluble fraction of the E. coli cell extract. The expression of the chimeric EcoCAc was confirmed by Western Blot (WB) analysis using an anti-His-Tag antibody ( Figure 3). As expected, analysing the E. coli cellular extract, the specific antibody for the His-Tag tail recognised the fusion protein as a band with a molecular weight of about 33.0 kDa. A subunit molecular mass of 32.4 kDa was calculated on the amino acid sequence translated from the chimeric gene encoding for the chimeric EcoCAc.

Heterologous expression and purification
The recombinant enzyme was purified to homogeneity using the affinity chromatography, as demonstrated by the SDS-Page analysis ( Figure 4). As a result, the electropherogram profile of EcoCAc showed a band at the same position identified in the western-blot analysis (33.0 kDa) under reducing conditions ( Figure 4).
The full sequence of the recombinant EcoCAc was also subjected to protonography, a powerful and elegant technique able to reveal, as a yellow band on the polyacrylamide gel, the production of ions (H þ ) developed during the CO 2 hydration reaction 63,65 . The protonography analysis demonstrated that the complete EcoCAc polypeptide chain was catalytically active. The protonogram showed a single hydratase activity band on the gel with a molecular weight of about 33.0 kDa, corresponding to the monomeric form of the chimeric EcoCAc ( Figure 5). This was not a surprise since the protonography analysis requires the elimination of SDS at the end of the electrophoretic run. Even if all the c-CAs are active as trimers, the yellow band appeared in the position of the inactive monomeric form because the SDS purging leads to the rearrangement of the c-CA monomers in the gel. As a result, EcoCAc correctly refolded and generated the active trimeric forms of the c-CA at the position of the monomer. This is described for other eukaryotic and prokaryotic CA classes, too 63

Determination of the kinetic parameters using the stoppedflow technique
The CO 2 hydratase activity of the soluble enzyme and its kinetic constants were determined using the stopped-flow technique ( Table 1). These results were compared with the kinetic parameters of the two mammalian a-CA isoforms (h CAI and h CAII), as   well as with the b-CA from the same microorganism and the a-, b-, and c-CAs from Vibrio cholerae ( Table 1). As shown in Table 1, EcoCAc is an excellent catalyst for the physiological CO 2 hydratase reaction to bicarbonate and protons, with a k cat of 5.7 Â 10 5 s À1 and catalytic efficiency (k cat /K M ) of 6.9 Â 10 6 M À1 s À1 . EcoCAc k cat was similar to those obtained for other bacterial CAs belonging to the b-or cclasses, as well as for the human isoform hCA I. Interestingly, the catalytic efficiency of EcoCAc resulted to be two orders of magnitude lower with respect to that of hCA II and one order with respect to VchCAc (same class of EcoCAc) and the other enzymes reported in Table  1. The investigation of the kinetic properties of the CAs is important because even if these enzymes belong to the same or different CA-classes the steric hindrance of the amino acid residues surrounding the catalytic pocket is responsible for the increase/ decrease of the parameters related to the affinity of the enzyme for the substrate (K M ). EcoCAc was also inhibited by the sulphonamide acetazolamide (K I ¼ 248 nM), a well-known pharmacological CA inhibitor ( Table 1). The acetazolamide resulted to be a very sensitive inhibitor of the human isoform h CAII (K I ¼12 nM) and the a-CA from Vibrio cholerae (K I ¼6.8 nM), wheras for the other CAs reported in Table 1 it was less effective, with K I s in the range of 227-473 nM. Thus, the K I variation can be attributed to the interaction and steric hindrance of the amino acid residues of the catalytic pocket interacting with the inhibitor. The structural differences, which affect the CA-classes or CAs belonging to the same class, highlight the possibility of designing specific and selective inhibitors for this superfamily of enzymes.

Sulphonamide inhibition profile
Among the CAIs, a library of 42 compounds, 1-24 and AAZ-EPA, represent an important group of simple aromatic/heterocyclic sulphonamides (including one sulfamate), which are able to inhibit the CA (Figure 6).
The series AAZ-EPA includes licenced drugs used for the following clinical treatments: glaucoma, epilepsy, idiopathic intracranial hypertension, diuretic, duodenal ulcers, migraine, Parkinson's disease, obesity, cancer, osteoarthritis, rheumatoid arthritis, diet, etc. [56][57][58] . Most of the sulphonamides bind in a tetrahedral geometry to the Zn(II) ion in the deprotonated state, establishing with the amino acid residues of the catalytic site an extended network of hydrogen bonds 18,19,22 , as well as the aromatic/heterocyclic part of the inhibitor interacts with the hydrophilic and hydrophobic residues of the catalytic cavity 18,19 .
Since CAs are considered a valuable target for impairing the microbe vitality or their virulence, the in vitro exploration of the EcoCAc inhibition profile with such inhibitors is crucial for obtaining potent and selective families of new pharmacological agents. New drugs are needed considering that the emergence arisen Figure 6. The 42 compounds used to study the EcoCAc inhibition profile. Forty-one sulphonamides and one sulfamate (TPM) were exploited. On the left, the series 1-24; on the right and grey, the clinically used drugs.
from the resistance to the existing antimicrobial medicines is one of the most severe problems afflicting the human community. Table 2 reports the inhibition profile of EcoCAc, which was compared with the inhibitory behaviour of hCA I, hCA II, and VchCAc reported earlier by our group 17,56,71 . From the data of The behaviour of EcoCAc is somewhat challenging to explain observing the different inhibition profiles and comparing them to the orthologous VchCAc and the two human isoforms (hCA I and hCA II). EcoCAc seems less or not inhibited by most of the substituted benzene-sulphonamides and clinically licenced drugs. However, considering that also the two human isoforms showed a sulphonamide inhibition pattern different from each other, as well as from the bacterial enzymes, it is reasonable to support the thesis concerning the synthesis of new drugs capable of interfering selectively with EcoCAc or VchCAc activity, avoiding the inhibition of the human CAs (a-class enzymes).

Conclusions
Escherichia coli is an opportunistic pathogen typically colonising the lower intestine of warm-blooded organisms [51][52][53][54] . In the present manuscript, we focussed our interest on the EcoCAc (c-CA) encoded by the E. coli genome since bacterial CAs are considered a valuable target for impairing the microbe vitality or the bacterial virulence. EcoCAc was cloned, expressed, purified, and investigated for its kinetic properties, the last not explored previously even if the three-dimensional structure was solved in 2012 (55). EcoCAc resulted in an excellent catalyst for the physiological CO 2 hydratase reaction to bicarbonate and protons, with a k cat of 5.7 Â 10 5 s À1 and catalytic efficiency (k cat /K M ) of 6.9 Â 10 6 M À1 s À1 . A broad range of substituted benzene-sulphonamides and clinically licenced drugs were used to determine the inhibition profile of EcoCAc, the ortholog enzyme VchCAc, and the possible off-targets hCA I and hCA II. Among the sulphonamides and the one sulfamate used as inhibitors, only one of them resulted in a K I lower than 100 nM. This is the case of the benzolamide (BZA) Table 2. Inhibition of hCA I, hCA II, EcoCAc and VchCAc with sulphonamides 1-24 and the clinically used drugs AAZ-EPA. whit a K I ¼ 94 nM. Generally, BZA is clinically used in the treatment of glaucoma. All the other inhibitors had K Is > 100 nM. Surprisingly, for most of the inhibitors used, EcoCAc showed 0.5 <K is < 0.5 mM, evidencing that enzyme form E. coli was less or not inhibited by most of the substituted benzene-sulphonamides and clinically licenced drugs. As a consequence, the difference in sulphonamide inhibition pattern of the two human isoforms, as well as the two orthologs bacterial enzyme fortifies the thesis that the synthesis of new drugs is capable of interfering selectively with EcoCAc or VchCAc activity, avoiding the inhibition of the human CAs (a-class enzymes).