Expression and characterization of a recombinant psychrophilic γ-carbonic anhydrase (NcoCA) identified in the genome of the Antarctic cyanobacteria belonging to the genus Nostoc

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the CO₂ hydration/dehydration reversible reaction: CO₂ + H₂O ⇄  + H⁺. Living organisms encode for at least six distinct genetic families of such catalyst, the α-, β-, γ-, δ-, ζ- and η-CAs. The main function of the CAs is to quickly process the CO₂ derived by metabolic processes in order to regulate acid-base homeostasis, connected to the production of protons (H⁺) and bicarbonate. Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources). It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average −1.9 °C all year round. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA, a γ-CA isolated from the Antarctic cyanobacterium Nostoc commune. This enzyme showed a higher catalytic efficiency at lower temperatures compared to mesophilic counterparts belonging to α-, β-, γ-classes, as well as a limited stability at moderate temperatures.

• Antarctic carbonic anhydrase
• carbonic anhydrase
• cyanobacteria
• hydratase activity
• metalloenzymes

Introduction

Carbon dioxide (CO₂) is an important molecule in all life processes and is generated in high amounts in all organisms, which developed catalysts for its transformation into protons and bicarbonates1–11. These catalysts are the enzymes known as carbonic anhydrases (CAs, EC 4.2.1.1) which catalyze the CO₂ hydration/dehydration reversible reaction: CO₂ + H₂O ⇄ + H⁺1^,3–6^,8^,12–19. Living organisms encode for at least six distinct genetic families of such catalyst, the α-, β-, γ-, δ-, ζ- and η-CAs3^,10^,15^,20–25. The main function of the CAs is to quickly process the CO₂ derived by metabolic processes in order to regulate acid–base homeostasis, connected to the production of protons (H⁺) and bicarbonate, also intervening in the secretion of electrolytes and biosynthetic processes in many cell types1–11. In fact, pH regulation is a tightly controlled process in most biochemical reactions due to the strong pH dependence of enzyme activity4^,26–47. Mesophilic CAs are considered among the most-active enzymes known, with a catalytic efficiency approaching the limit of diffusion-controlled processes24^,48–76.

It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average −1.9 °C all year round77–84. Antarctic organisms have evolved several strategies to overcome the effect of low temperatures on catalytic efficiency. They can follow different strategies to achieve cold adaptation at the metabolic level, such as77^,78^,80^,81^,83^,84: (a) the increase of enzyme production in order to compensate for reduced kinetic efficiency; (b) the expression of enzymes with relatively higher substrate turnover and the capacity to maintain ligand-binding properties at low temperatures; and (c) cold-adapted enzymes evolved higher K_m values (at any measured temperature) compared to their warm-adapted orthologous. Most of these strategies involve alteration of various specific features of the enzyme, in particular functionality and stability77^,78^,80^,81^,85–88. They also represent the major hallmarks of psychrophily and have been shown to be specific from organism to organism. Probably, cold adaptation traits do not follow specific set patterns and perhaps are currently in a state of continuous evolution influenced by the environmental conditions, such as low temperature, high salinity.

Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources), which are active at physiological temperatures of around 37 °C. Recently, our group studied CAs from microorganisms living in extreme habitats, such as thermophiles living at temperatures ranging from 70 to 110 °C1^,9^,10^,25^,89–95. These enzymes were highly active for the hydration of CO₂ to bicarbonate and protons and possessed an inhibition profile (with anions and sulfonamides) very similar to those of the mesophilic human (h) cytosolic isoform hCA II. In this context, we started to study CAs from psychrophiles96^,97. Antarctica can be considered as a huge natural laboratory and many scientists share the common interest of elucidating the evolutionary mechanisms developed by Antarctic organisms in order to adapt to this very extreme habitat77^,78. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures77^,78. Recently, we studied the catalytic activity of a γ-CA, named NcoCA, identified in the genome of the diazotrophic cyanobacteria belonging to the genus Nostoc, isolated from the freshwaters of Antarctic lakes, which form colonies composed of filaments of moniliform cells in a gelatinous sheath96^,97. The enzyme showed a significant catalytic activity for the physiologic reaction, CO₂ hydration to bicarbonate and protons, with a k_cat of 9.5 × 10⁵s⁻¹ and a k_cat/K_m of 8.3 × 10⁷M⁻¹ s⁻¹, being the most catalytically efficient γ-CA investigated so far. Our groups also carried out an extensive study using inorganic/organic anions and aromatic/heterocyclic sulfonamides inhibitors of NcoCA. The best NcoCA inhibitors detected so far were diethyldithiocarbamate (K_I of 0.80 mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (K_I in the range of 70–90 mM); while inhibitors belonging to sulfonylated sulfanilamide derivatives possessing elongated molecules, aminobenzolamide, acetazolamide, benzolamide, dorzolamide, brinzolamide and topiramate, showed inhibition constants in the range of 40.3–92.3 nM. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA.

Materials and methods

Gene identification

The identification of Nostoc γ-CA (NcoCA) was performed at the link http://www.ncbi.nlm.nih.gov/genome98 using in the keyword fields the word: “Nostoc”. The γ-CA of Nostoc sp. was identified running the “BLAST” program98 using as query sequences CAM or CAMH (γ-CAs from Methanosarcina thermophila)99^,100.

Construct preparation, protein expression and purification

Our groups designed the synthetic NcoCA DNA sequence and included the restriction enzyme NdeI and XhoI at the 5′ and 3′ end of the NcoCA gene, respectively. The gene was optionally optimized using the Gene Optimizer (Life-Technologies, Carlsbad, CA) software for maximum protein production. The GeneArt Company, specialized in gene synthesis, produced the aforementioned synthetic gene, which was provided in a standard vector (sub-cloning vector). The plasmid was amplified into E. coli DH5 α cells. The NcoCA DNA fragments were separated on 1% agarose gel. The recovered NcoCA gene and the linearized expression vector (pET15-b) were ligated by T4 DNA ligase to form the expression vector pET15-b/NcoCA. At the N-terminal sequence of NcoCA, pET15-b/NcoCA construct expressed a short polypeptide containing six histidines to facilitate the purification of the target protein. In order to confirm the integrity of the NcoCA gene and the fact that no errors occurred at the ligation sites, the vector containing the fragment was sequenced. Arctic Express competent cells (Agilent Technologies, La Jolla, CA) were transformed with pET15-b/NcoCA expression vector. Several transformant colonies were picked and grown overnight in LB containing 20 mg/ml of gentamicin and 100 mg/ml of ampicillin for selecting the expression plasmid at 37 °C. Then, cells were grown without antibiotic selection at 30 °C and induced at an A₆₀₀ of 0.6–1 with 1 mM isopropylthiogalactopyranoside (IPTG). Zn(SO₄) was added after 30 min. After additional growth for 16 h at 20 °C, cells were harvested, resuspended in 10 mM buffer Tris/HCl, pH 8.3 and disrupted by sonication at 4 °C. Following centrifugation, soluble protein extract was loaded onto a His-select HF nickel affinity gel column (GE Healthcare, Little Chalfont, Buckinghamshire, UK), then washed with five column volumes with His binding buffer (20 mM NaH₂PO₄, 500 mM NaCl, 20 mM Imidazole, pH 8). The His-tagged protein was eluted from the column with elution buffer (20 mM NaH₂PO₄, 500 mM NaCl, 250 mM Imidazole, pH 8). The purified recombinant NcoCA was dialyzed over-night against 10 mM buffer Tris/HCl, pH 8.3. At this stage of purification the enzyme was at least 90% pure and the obtained recovery was of 2 mg of the recombinant Antarctic γ-CA.

Sequence analysis

Multialignment of amino acid sequences was performed using the program MUSCLE (MUltiple Sequence Comparison by Log- Expectation), a new computer program for creating multiple alignments of protein sequence101.

SDS-PAGE

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli using 12% gels102.

Protonography

Briefly, wells of 12% SDS-gel were loaded with bCA, NcoCA and PgiCA mixed with loading buffer without 2-mercaptoethanol and without boiling the samples, in order to avoid protein denaturation. The gel was run at 180 V until the dye front ran off the gel. Following the electrophoresis, the 12% SDS-gel was subject to protonography to detect the PgiCA and NcoCA hydratase activity on the gel as described by Capasso and coworkers22^,103^,104.

Assay for carbonic anhydrase with CO₂ as substrate

Colorimetric method

CA activity assay was a modification of the procedure described by Chirica et al.105. The assay was based on monitoring the pH variation due to the catalyzed conversion of CO₂ to bicarbonate. Bromothymol blue was used as the indicator of pH variation. The assay was performed at 0 °C adding 1.0 mL ice-cold CO₂-saturated water to 1.0 mL mixtures of 25 mM Tris-SO₄ buffer containing different amounts of the enzyme. The CO₂-satured solution was prepared by bubbling CO₂ into 100 mL of distilled water for approximately 3 h. The CO₂ solution was chilled in an ice-water bath. To test the activity of the enzyme, 1 mL of 25 mM Tris, pH 8.3, containing bromothymol blue as a dye (to give a distinct and visible blue color) was added to two test tubes chilled in an ice bath. A volume of 10 to 50 μL of the enzyme solution (e.g. cell extract or purified enzyme) was added to one tube, and an equivalent amount of buffer was added to the second tube as control. One milliliter of CO₂ solution was added very quickly and simultaneously a stopwatch was started. The time required for the solution to change from blue to yellow was recorded (transition point of bromothymol blue is pH 6–7.6). The production of hydrogen ions during the CO₂ hydration reaction lowers the pH of the solution until the color transition point of the dye is reached. The time required for the color change is inversely related to the quantity of carbonic anhydrase present in the sample. Detecting the color change is somewhat subjective, but the error for triple measurements was in the range of 0–1 s difference for the catalyzed reaction. Wilbur–Anderson units were calculated according to the following definition: One Wilbur–Anderson unit (WAU) of activity is defined as (T0 − T)/ T, where T0 (uncatalyzed reaction) and T (catalyzed reaction) are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye in a control buffer and in the presence of enzyme, respectively.

Sopped-flow technique

An applied photophysics stopped-flow instrument was used for assaying the CA catalyzed CO₂ hydration activity106. Phenol red (at 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm with 10 mM Hepes (pH 7.5) as buffer and 0.1 M NaClO₄ (for maintaining constant ionic strength), at 20 °C, following the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s (the uncatalyzed reaction needs around 60–100 s in the assay conditions, whereas the catalyzed ones are of around 6–10 s). The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Enzyme concentrations in the assay system were about 10 nM for all the enzymes considered in the present study.

Effect of temperature on the NcoCA stability

To compare the stability of NcoCA and PgiCA at different temperatures, enzyme solutions at the concentration of 3 μg/mL in 10 mM Tris/HCl, pH 8.3 were incubated at 4, 10, 25, 40, 50 and 60 °C for different times (10, 20, and 60 min). Enzyme aliquots (30 ng) were withdrawn at appropriate times and the residual activity was measured at 0 °C using CO₂ as substrate.

PgiCA (γ-CA) preparation

PgiCA protein was prepared as described by Del Prete et al.16.

Results

Sequence analysis

We aligned the amino acid sequence of NcoCA with the amino acid sequence of a mesophilic γ-CA, PgiCA, to try to identify unique features of this Antarctic enzyme (Figure 1). The open reading frame of the CA gene identified in the genome of the Antarctic cyanobacterium encodes for polypeptide chain of 188 amino acid residues displaying 48% identity when compared with PgiCA (γ-CA from the bacterium Porphyromonas gingivalis). It may be observed that similar to the other investigated γ-CA, NcoCA has the conserved three His ligands, which coordinate the Zn(II) ion crucial for catalysis (His81, 117 and 122, CAM numbering system99^,100).

Figure 1. Amino acid sequence alignment of NcoCA and PgiCA (γ-CA). The metal ion ligands (His81, Hist117 and His122) are indicated in bold. The multialignment was performed with the program Muscle. Numbering system of the most known γ-CA, named CAM, was used 99^,100. The asterisk (*) indicates identity at all aligned positions; the symbol (:) relates to conserved substitutions, while (.) means that semi-conserved substitutions are observed.

Enzyme purification

To study the biochemical properties of this psychrophilic enzyme, the recombinant NcoCA was isolated and purified to homogeneity at room temperature from E. coli (DE3) cell extract. Most of the CA activity was recovered in the soluble fraction of cell extract obtained after sonication and centrifugation. Using the affinity column (His-select HF Nickel affinity gel), NcoCA was purified to an apparent homogeneity of 90%, as indicated by SDS-PAGE (Figure 2, lane 1). The apparent molecular weight estimated by SDS-PAGE of the purified His-tag NcoCA was 22.5 kDA. A subunit molecular mass of 22.0 kDa was calculated on the basis of the amino acid sequence translated from the NcoCA gene containing at the 5′ the His-tag nucleotide sequence. As described in the literature, tri-dimensional γ-CA structure revealed a trimeric arrangement for this class of CAs. Moreover, each monomer resembles an equilateral triangle stabilized by H bonds, salt bridges and hydrophobic interactions99^,100^,107. The SDS–PAGE of NcoCA showed two bands: one corresponding to a monomer with an apparent molecular weight of 22 kDa; while the second band showed an apparent molecular weight of 55 kDa, which corresponded to the trimeric form of the γ-CA, NcoCA. The trimer contains three active sites and each monomer contributes His residues located on the surface, at the interface between two adjacent monomers, to coordinate the Zn(II) ion crucial for catalysis99^,100^,107–109.

Figure 2. SDS-PAGE of the recombinant NcoCA purified from E. coli cells. Legend: Lane 1, molecular markers; Lane 2, purified NcoCA from His-tag affinity column. NcoCA showed two bands: one corresponding to a monomer with an apparent molecular weight of 22 kDa; the second band showed an apparent molecular weight of 55 kDa.

Biochemical characterization

Protonography

The hydratase activity of NcoCA on the SDS–PAGE gel was investigated and compared with that obtained for other CA classes, such as PgiCA (γ-CA) and bovine (b) bCA (α-CA). The gels were run under denaturing and non-reducing conditions. The protonogram showed in Figure 3 was obtained loading on the on the SDS–PAGE samples of NcoCA, PgiCA and bCA at 10 μg/well. As described in the experimental section, the protonography is based on monitoring the pH variation in the gel due to the CA-catalyzed conversion of CO₂ to bicarbonate and protons. The protonogram is then stained with bromothymol blue, which is a widely used pH indicator. This dye appears blue in its deprotonated form, while its color changes to yellow in the protonated form. Thus, the production of ions (H⁺) during the CO₂ hydration reaction, due to the CA hydratase activity, lowers the pH of the solution until the color transition point of the dye is reached, that is, at pH 6.8. It is interesting to note that NcoCA showed a different behavior on the protonogram respect to the other γ-CA, PgiCA (Figure 3). The protonogram showed that PgiCA is present in two oligomeric state, monomer and trimer, while NcoCA showed only a hydratase activity band at the position corresponding to the molecular weight of its monomer. This suggests that probably the Antarctic enzyme reacted differently to the SDS present in the loading buffer with respect to the mesophilic enzyme, PgiCA. The α-CA, bCA, showed only one hydratase activity band (28 kDa) because its oligomeric state is monomeric22^,103^,104. It was possible to detect the hydratase activity of the inactive monomer of the γ-CA because, as described previously, following the electrophoresis, SDS was removed from the gel. This procedure potentially led to the rearrangement of γ-CA monomers in the gel, reconstituting thus the active trimeric form of the enzyme. The final result was the presence of a yellow band at the position of a γ-CA monomer22^,103^,104.

Figure 3. Protonogram obtained using bCA (α-CA) and two γ-CAs: the Antarctic NcoCA and mesophilic PgiCA. The yellow bands correspond to the CA position on the gel responsible for the drop of pH from 8.2 to the transition point of the dye in the control buffer. Incubation time was of 20 s. NcoCA showed only one band (22 kDa), while PgiCA was present in two oligomeric states, the monomeric (22 kDa) and the trimeric (55 kDa) forms (see text for details).

Kinetic properties

A stopped-flow CO₂ hydrase assay has been used to measure the catalytic activity of NcoCA. Table 1 shows a comparison of the kinetic parameters for the CO₂ hydration reaction catalyzed by NcoCA, hCAI and II (α-CA from Homo sapiens, isoform I and II, respectively), Can2 (β-CA from Cryptococcus neoformans), PgiCA (γ-CA from the bacterium Porphyromonas gingivalis) and CAM, (γ-CAs from Methanosarcina thermophila). It may be observed that NcoCA showed kinetic parameters about two times higher than the other γ-CA reported in Table 1 (PgiCA). Moreover, the inhibition constant for acetazolamide (AZA) is similar to that obtained for the CAM.

Table 1. Kinetic parameters for the CO₂ hydration reaction catalyzed by the α-, β- and γ-CAs at 20 °C and pH 7.5 in 10 mM HEPES buffer and 20 mM Na₂SO₄, measured at 20 °C, pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO₄77.

Enzyme	Activity level	Class	kcat (s^−1)	kcat/Km (M^−1× s^−1)	KI (acetazolamide) (nM)
hCA I	moderate	α	2.0 × 10^5	5.0 × 10^7	250
hCA II	very high	α	1.4 × 10^6	1.5 × 10^8	12
Can2	moderate	β	3.9 × 10^5	4.3 × 10^7	10.5
CAM	low	γ	6.1 × 10^4	8.7 × 10^5	63
PgiCA	moderate	γ	4.1 × 10^5	5.4 × 10^7	324
NcoCA	high	γ	9.5 × 10^5	8.3 × 10^7	75.8

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

Temperature effect on the stabilities of VchCA and bCA II

Stabilities of NcoCA and PgiCA were compared at the temperatures of 4, 10, 30, 40, 50, 60 °C (Figure 4). After 10, 20 and 60 min of incubation time, the activities of NcoCA and PgiCA were determined using CO₂ as substrate. As indicated in Figure 4, after 60 min of incubation the activity of the Antarctic enzyme decreased to 20% at temperatures higher than 45 °C, and was completely inactivated at 60 °C. Otherwise, PgiCA, the mesophilic enzyme, retained its activity up to 60 °C (Figure 4) at all incubation times. It is interesting to note that the Antarctic enzyme showed an optimal activity at low temperature (4–20 °C) compared to the mesophilic counterpart (PgiCA). This behavior was observed at all the incubation times (Figure 4) reflecting a typical feature of the cold-adapted enzyme.

Figure 4. Thermostability of NcoCA and PgiCA. The enzymes were incubated for 10 (A), 20 (B) and 60 (C) min at the temperatures of 4, 10, 30, 40, 50, and 60 °C. Enzyme activity were assayed using CO₂ as substrate.

Discussion and conclusions

Antarctica can be considered as a huge natural laboratory for elucidating the evolutionary mechanisms developed by organisms, which were able to live and thrive in such an extreme habitat. Changes at the genetic level are relevant at the level of the molecular structure. For example, they might preserve the structure and function of an enzyme even though the molecule has to work in a completely different environment, i.e. the Antarctic sea where temperatures average −1.9 °C all year round. In general, cold-adapted enzymes show higher catalytic efficiency at low temperatures compared to their mesophilic counterparts. This characteristic is mainly due to the higher flexibility of the protein structure at low temperatures. Indeed, the tridimensional structures of the cold-adapted enzymes show a lower number of stabilizing factors, such as salt bonds, hydrogen bonds, hydrophobic interactions and a lower content of proline and arginine residues than their mesophilic counterparts77–80. For this reason, the study of CAs from Antarctic species is of interest, especially in relation to the strategies adopted by these organisms to achieve a normal level of catalytic activity at temperatures below that of the mesophilic species. The present paper describes the biochemical characterization of a γ-CA, named NcoCA, identified in the genome of the diazotrophic cyanobacteria belonging to the genus Nostoc, isolated from the freshwaters of Antarctic lakes, and confirms that all these adaptations to cold mentioned above are also present for this type of enzymes.

From the biochemical features of the purified enzyme and from the amino acid sequence analysis, NcoCA described in the present paper is a CA belonging to the γ-CA family. Enzyme from cold-adapted species commonly have an optimal activity at relatively low temperature; above this value, the loss of activity is usually attributed to thermal inactivation as a consequence of the increased structural flexibility of the protein. In fact, cold-adapted enzymes are typified by: (i) a higher catalytic efficiency than their mesophilic counterpart over a temperature range from 0 to 30 °C, and (ii) limited stability at moderate temperatures. It is immediately apparent that NcoCA meets the requirements of criterion (i) (at 25 °C NcoCA_kcat/Km = 8.3 × 10⁷, PgiCA_kcat/Km = 5.4 × 10⁷). Intriguingly, at 25 °C NcoCA_kcat/Km is two order of magnitude higher than that determined for the γ-CA from the thermophilic bacteria Methanosarcina thermophila (CAM_kcat/Km = 8.7 × 10⁵). Even more clearly, it is evident that the Antarctic CA (NcoCA) completely meets the requirement of criteria (ii) (Figure 4). NcoCA thermolability decreases with the increase of temperature (Figure 4). The determinants of protein stability include structural factors, hydrophobic effects, and mainly weak interactions between atoms of the protein structure110. Cold-adapted enzyme are characterized by clustering of glycine residues, providing local mobility; the disappearance of proline residues in loops, enhancing chain flexibility between secondary structures; a reduction in arginine residues which are capable of forming multiple salt bridges and H-bonds as well as a lower number of ion pairs, aromatic interactions, or H-bonds, compared to mesophilic enzymes110. Comparison of the amino acid sequence of NcoCA with those of the mesophilic (PgiCA) or thermophilic (CAM) counterparts is far from being exhaustive, as it does not take into account all the details aforementioned. But the exploitation of recombinant DNA technology would allow the production of sufficient amount of NcoCA for structural studies to be undertaken by X-ray crystallography. In conclusion, the elucidation of the NcoCA structure, together with site-specific mutagenesis coupled with kinetic analysis, may provide a rational explanation for the thermolability displayed by the Antarctic CA with respect to the mesophilic and thermophilic such enzymes.

Declaration of interest

We thank the Distinguished Scientist Fellowship Program (DSFP) at KSU for funding this project. This work was also financed in part by an FP7 EU project (Dynano). The authors report no conflicts of interest.