INTRODUCTION

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Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration of CO₂ (CO₂ + H₂O  HCO₃ + H⁺). In eukaryotes, the enzyme participates in various physiological functions, which include the interconversion of CO₂ and HCO₃ during photosynthesis and intermediary metabolism, facilitated diffusion of CO₂, pH homeostasis, and ion transport (5, 45). Carbonic anhydrase is nearly ubquitous in highly evolved eukaryotes, but until recently the enzyme was thought to play a minor role in prokaryotic physiology (48). All carbonic anhydrases are divided into three distinct classes (, , and ) that have no sequence homology and evolved independently (22). Carbonic anhydrases from mammals (including the 10 active human isoforms) (22, 39) together with the two periplasmic enzymes from the unicellular green alga Chlamydomonas reinhardtii (16, 17) belong to the  class. The  class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants (22). Four prokaryotic carbonic anhydrases from species in the Bacteria domain, two belonging to the  class and two belonging to the  class, have been described (13, 20, 49, 50). The only carbonic anhydrase purified from an archaeon, Methanosarcina thermophila, is decidedly distinct from the  and  classes and is the prototype of a novel class ( class) (2).

Crystal structures have been determined for five isozymes (CA I to CA V) of the monomeric human carbonic anhydrase (9, 14, 21, 28, 51) and the Neisseria gonorrhoeae enzyme (25) belonging to the  class. The overall folds of these monomeric enzymes are highly similar, with a 10-stranded, mainly antiparallel, -sheet as the dominating structure. The catalytically active zinc is ligated to three histidine residues, with a water molecule acting as a fourth ligand. The structure of the prototype  carbonic anhydrase from M. thermophila has recently been solved and found to be entirely different from those of the -class enzymes (31). The enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers. The zinc is coordinated in a tetrahedral geometry with three histidines and two to three putative water molecules serving as ligands (1). The main secondary structures are several parallel -sheets forming a left-handed -helix. Although a three-dimensional structure has yet to be determined for a  carbonic anhydrase, extended X-ray absorption fine structure and circular dichroism of the plant enzyme indicate that the zinc coordination and overall structure are quite different from those for either the - or -class enzymes (10, 42).

Methanoarchaea, the largest group within the Archaea domain, obtain energy for growth by either the production of methane from the reduction of CO₂, utilizing H₂ or formate as electron donors, or the conversion of the methyl groups of acetate, methanol, or methylamines to methane (8, 15). To date, the only characterized carbonic anhydrase from the Archaea domain is the  carbonic anhydrase (Cam) from the methanoarchaeon M. thermophila, which can convert the methyl group of acetate, methanol, or methylated amines to methane. Western blot analysis indicates that Cam is expressed predominantly during growth on acetate, suggesting that this carbonic anhydrase is important for acetotrophic growth (3). Cam appears to be located outside the cell, and it has been proposed that it might be required for a CH₃CO₂/H⁺ symport system or for efficient removal of cytoplasmically produced CO₂ during growth on acetate (2).

Here we report on the initial characterization of a  carbonic anhydrase from Methanobacterium thermoautotrophicum H, the first carbonic anhydrase from a CO₂-reducing methanoarchaeon in the Archaea domain. Our results show that  carbonic anhydrases occur not only in the Archaea domain but also in thermophilic chemolithoautotrophs, species that represent some of the deepest branches of the universal tree of life (53).

	MATERIALS AND METHODS

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Cloning and hyperexpression of the cab gene in Escherichia coli. Two oligonucleotides (primers MBTCA1 [5'-GGTTTGTTACCATGGTTATTAAAG-3'; partially corresponding to the amino terminus of Cab] and MBTCA2 [5'-CGTAGAGGATCCTTCAG-3'; partially corresponding to the carboxy terminus of Cab]), 100 ng of M. thermoautotrophicum H genomic DNA, and the GeneAmp DNA amplification kit (Perkin-Elmer Cetus) were used to amplify the genomic region containing the cab gene. The amplification generated NcoI and BamHI restriction sites on the ends of the amplified product. The product was digested with NcoI and BamHI and cloned into the appropriately digested pET16b (Novagen) to generate plasmid pMBTCA13. E. coli BL21(DE3) competent cells were transformed with pMBTCA13, grown at 37°C in Luria-Bertani broth containing 100 µg of ampicillin per ml, and induced with 27.8 mM lactose and 0.5 mM zinc sulfate (final concentration) at an A₆₀₀ of 0.8. After an induction of 3 h at 37°C, the cells were harvested by centrifugation and stored at 70°C.

Purification of the heterologously produced Cab. Carbonic anhydrase activity was measured at room temperature, using a modification of the electrometric method of Wilbur and Anderson as described previously (58). Protein concentrations were determined by the Bradford method (11), using Bio-Rad dye reagent and bovine serum albumin (Sigma) as the standard. Thawed cell paste (10 g [wet weight]) was suspended in 20 ml of buffer A (50 mM potassium phosphate [pH 6.8]) and passed twice through a chilled French pressure cell at 138 MPa. The cell lysate was centrifuged at 20,000 × g for 20 min to remove cell debris. The supernatant solution was recentrifuged at 100,000 × g for 2 h. The cell extract was then heated at 65°C for 20 min and centrifuged at 20,000 × g for 15 min. The supernatant was loaded onto a Mono Q 10/10 anion-exchange column (Pharmacia) equilibrated with buffer A. After a 30-ml wash, the column was developed with a 100-ml linear gradient from 0 to 1 M KCl. Fractions containing active enzyme were pooled, desalted, and again run on a Mono Q column. The enzyme eluted between 460 and 520 mM KCl. The active fractions were pooled and stored at 20°C.

Esterase activity. Activity for p-nitrophenylacetate hydrolysis was determined at 25°C, using a modification of the method of Armstrong et al. (4). The reaction mixture (1.35 ml) contained freshly prepared 3 mM p-nitrophenylacetate in acetone. The uncatalyzed rate of the reaction was determined by adding 0.15 ml of 100 mM potassium phosphate (pH 7.0) and recording the change in A₃₄₈ per min ( = 5,000 M¹ cm¹). After 2 min, 15 µl of enzyme solution was added, and the catalyzed reaction was monitored for an additional 3 min.

Molecular mass determination. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (33), using 12% gels. The native molecular mass was determined by gel filtration chromatography, using a Superose 12 gel filtration column (Pharmacia) calibrated with bovine milk -lactalbumin (14.2 kDa), bovine erythrocyte carbonic anhydrase (29 kDa), chicken egg albumin (45 kDa), bovine serum albumin (66 kDa; dimer, 132 kDa), and urease (trimer, 272 kDa; hexamer, 545 kDa). Protein samples (0.5 ml) were loaded onto the column pre-equilibrated with buffer A containing 150 mM KCl, and the column was developed with a flow rate of 0.4 ml/min.

Metals analysis. A comprehensive metals analysis (20 elements) was carried out by inductively coupled plasma atomic emission spectroscopy, using a Jarrel Ash Plasma Comp 750 instrument at the Chemical Analysis Laboratory, University of Georgia. All solutions were prepared in plasticware, using deionized water (18 ) from a Barnstead-Thermolyne deionization system. Dialysis tubing (Spectrum) was treated and buffers were made metal free by batch treatment with Chelex 100 (Bio-Rad) (3, 24). Samples of two independent enzyme preparations were first concentrated in dialysis tubing (3.5-kDa cutoff) embedded in dry polyethylene glycol (M_r = 8000; Sigma) at 4°C before dialysis against 2 liters of metal-free buffer (20 mM potassium phosphate [pH 6.8]) for 20 h at 4°C. A sample of each enzyme preparation along with a sample of buffer alone were analyzed for metals content. Protein concentrations were determined by the biuret method (18), with bovine serum albumin (Sigma) as the standard. The results with the biuret method indicated that the Bradford method underestimated the carbonic anhydrase protein concentration by a factor of 2.

Steady-state kinetics. Initial rates of CO₂ hydration were determined by stopped-flow spectroscopy (KinTek stopped-flow apparatus; State College, Pa.) at 25°C, using the changing pH indicator method (29). Saturated solutions of CO₂ were prepared by bubbling CO₂ into distilled, deionized water at 25°C, and the CO₂ concentration was varied from 6 to 24 mM by mixing with an appropriate volume of N₂-saturated water. CO₂ hydration activity was determined at pH 8.5 with a final Cab concentration of 2 µM. The absorbance change was measured at 578 nm in a final buffer concentration of 50 mM TAPS (N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid; pK_a 8.4)-28.6 mM Na₂SO₄-48.6 µM m-cresol purple. The steady-state parameters k_cat and k_cat/K_m and their standard errors were determined by fitting the observed initial rates (corrected for the uncatalyzed reaction) to the Michaelis-Menten equation, using the Kaleidagraph program (Synergy Software, Reading, Pa.).

Western blot analysis. M. thermoautotrophicum H cells were suspended in 50 mM potassium phosphate (pH 6.8) and passed twice through a chilled French pressure cell at 138 MPa, and the cell extract was collected following centrifugation at 20,000 × g for 20 min to remove unbroken cells. Polyclonal antibodies directed against the purified heterologously produced Cab were raised in New Zealand White rabbits (Cocalico Biological Corp., Reamstown, Pa.). Cell extract proteins were separated by SDS-PAGE on 12.0% gels and electrotransferred to a polyvinylidene membrane (Immobilon; Millipore) as recommended (Bio-Rad). Additional protein sites were blocked by incubating the membrane in 10 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 0.05% Tween 20, and 10% evaporated milk. Primary antisera raised to Anabaena strain PCC7120 carbonic anhydrase ( type) (50), the M. thermoautotrophicum H Cab ( type), and the M. thermophila Cam ( type) (3) were used at dilutions of 1:10,000, 1:5,000, and 1:10,000, respectively. A 1:7,500 dilution of anti-rabbit immunoglobulin G-alkaline phosphatase conjugate was used. The antibody-antigen complex was detected with 5-bromo-4-chloro-3-indolylphosphate and 4-nitroblue tetrazolium chloride as recommended by the supplier (Boehringer Mannheim Biochemicals).

Materials. All chemicals were of reagent grade and purchased from Sigma or Fisher Scientific. Highly purified human carbonic anhydrase was obtained from Sigma. T4 DNA ligase and restriction enzymes were purchased from New England Biolabs. Gel filtration and SDS-PAGE molecular mass standards were from Sigma. Oligonucleotide primers were obtained from and sequencing was performed at the Nucleic Acid Facility, Pennsylvania State University.

	RESULTS AND DISCUSSION

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Heterologous production and purification of Cab. Recently, genome sequencing (41, 46) of the thermophilic, obligately chemolithoautotrophic methanoarchaeon M. thermoautotrophicum H revealed an open reading frame (ORF) with a deduced sequence 34.3% identical to that of CynT, the  carbonic anhydrase of E. coli (20). The gene, designated cab (carbonic anhydrase beta), was PCR amplified and cloned into the pET16b vector to produce plasmid pMBTCA13 and then expressed in E. coli by using the T7 promoter/polymerase expression system (54, 55). Greater than 95% of the carbonic anhydrase activity was recovered in the soluble fraction after ultracentrifugation of the E. coli cell extract for 2 h at 100,000 × g. By taking advantage of the thermal stability of Cab, a major purification step was the incubation of the high-speed (ultracentrifuge) soluble supernatant at 65°C for 15 min followed by centrifugation to remove the denatured E. coli proteins. The heterologously produced enzyme was purified 13-fold to apparent homogeneity (Table 1), as indicated by a single polypeptide band after SDS-PAGE (Fig. 1).

View larger version (73K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of heterologously produced Cab at various steps during purification from E. coli. Lane 1, 20 µg of cell extract protein from E. coli carrying pMBTCA13 prior to induction; lane 2, 20 µg of cell extract protein from E. coli carrying pMBTCA13 3 h after induction; lane 3, 100 µg of cell extract protein after centrifugation at 100,000 × g for 2 h; lane 4, 16 µg of protein after incubation at 65°C for 15 and centrifugation at 20,000 × g for 20 min; lane 5, 7 µg of protein from the first Mono Q step; lane 6, 7 µg of protein from the second Mono Q step; lane 7, 14 µg of protein from the second Mono Q step. Positions of molecular mass markers are shown on the left in kilodaltons.

Biochemical characterization. (i) Thermostability. The activity of Cab was stable when the enzyme was incubated for 15 min at temperatures up to 75°C (Fig. 2). This is not surprising since the optimal growth temperature for M. thermoautotrophicum H is between 65 and 75°C (8). Little activity was recovered when the enzyme was incubated at temperatures of 90°C or higher. Thus, Cab is the most thermostable carbonic anhydrase yet characterized.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Thermostability of Cab. Cab was incubated for 15 min at the indicated temperatures and cooled on ice, and activity was determined at 25°C by the electrometric method (58). Activity is represented as a percentage of the activity of a sample maintained on ice throughout the experiment (38.5 U/mg).

(ii) Inhibition. Iodide, nitrate, and azide are potent inhibitors of Cab (IC₅₀ [concentrations of inhibitor resulting in 50% inhibition of enzyme activity, determined by a semilogarithmic plot of percentage of inhibition versus logarithmic concentration of inhibitor] of 1.0, 1.5, and 2.1 mM, respectively); however, chloride and sulfate, which inhibit plant  carbonic anhydrases, had no effect on the activity of Cab (47). The insensitivity to chloride and sulfate suggests a fundamental difference in the active sites of Cab and the plant enzymes. The effect of these anions on the two other known prokaryotic  carbonic anhydrases has not been reported. The insensitivity to chloride and sulfate is also observed for Cam from M. thermophila, the only other known methanoarchaeal carbonic anhydrase (2, 3).

(iii) Esterase activity. Some carbonic anhydrases belonging to the  family catalyze the reversible hydrolysis of esters. With p-nitrophenylacetate as a substrate, commercially available human CA II showed an esterase activity of 38.2 mol of p-nitrophenylacetate per min per mol of enzyme. In contrast, Cab showed no detectable esterase activity (<0.01 mol of p-nitrophenylacetate per min per mol of enzyme); thus, Cab joins other carbonic anhydrases from the  class in lacking any detectable esterase activity (20, 30). Cam, the only characterized carbonic anhydrase of the  class, also lacks an esterase activity (3).

(iv) Subunit composition. A subunit molecular mass of 21 kDa was estimated by SDS-PAGE (Fig. 1). A subunit molecular mass of 18.9 kDa was calculated on the basis of the amino acid composition deduced from the nucleotide sequence. Native gel filtration chromatography of Cab gave an estimated molecular mass of 90 kDa. Given a calculated subunit molecular mass of 18.9 kDa, these results suggest that the native enzyme is a tetramer.

(v) Metals analysis. The carbonic anhydrases from the , , and  classes contain one zinc per enzyme subunit and are thought to have a common zinc hydroxide mechanism for catalysis. A comprehensive metals analysis (20 elements) of Cab was performed by plasma emission spectroscopy using two independent enzyme preparations with similar specific activities. Since the Bradford assay was found to underestimate the concentration of Cab by a factor of 2, the biuret assay was used to determine protein concentrations for the metals analysis. The analysis revealed 1.01 and 0.97 Zn per subunit of Cab for preparations I and II, respectively, suggesting Cab contains one zinc per subunit.

Kinetic properties of Cab. Kinetic parameters for the CO₂ hydration activity were measured at 25°C in a stopped-flow spectrophotometer. The values for Cab are presented in Table 2 along with the values for the kinetically characterized -class carbonic anhydrases as well as those for the only two kinetically characterized prokaryotic enzymes besides Cab, the prototypical -class methanoarchaeal enzyme and the -class neisserial enzyme. The K_m value for CO₂ is similar to those of the characterized enzymes of the  class; however, the k_cat for Cab is lower than those for the carbonic anhydrases isolated from higher plants (6, 34) and the unicellular green alga Coccomyxa sp. (23) (Table 2). One possible reason for the greater than 10-fold difference in k_cat and in the catalytic efficiency (k_cat/K_m) may be that the assay temperature was more than 40°C below the optimal growth temperature of M. thermoautotrophicum, which is between 65 and 75°C. The optimal temperature for Cab activity is expected to be near the growth temperature; however, the decreased solubility of CO₂ at these temperatures under atmospheric pressure precludes the determination of accurate kinetic parameters above 25°C.

Alignment of -class carbonic anhydrases. On the basis of amino acid sequence comparisons, carbonic anhydrases belong to three genetically distinct classes (, , and ) that evolved independently (22). We had previously identified 51 ORFs with deduced sequences having significant identity and similarity to the sequences of Cab from M. thermoautotrophicum H by Blastp and tBLASTn searches of the nonredundant sequence database at the National Center for Biotechnology Information and the finished and unfinished microbial genome sequences (48). Of the 26 ORFs identified from species of the Bacteria and Archaea domains, only 2 are known to encode documented carbonic anhydrases (20, 49). Distinct from all other  carbonic anhydrase sequences, the plant sequences form two monophyletic groups representing monocotyledons and dicotyledons. The remaining sequences form four clades, and Cab is found in one of the two clades composed exclusively of prokaryotic sequences.

View larger version (77K): [in this window] [in a new window]   FIG. 3.   Comparison of the deduced sequence of Cab with the deduced sequences of putative carbonic anhydrases. The deduced amino acid sequences were aligned by using ClustalX (56). Abbreviations and GenBank accession numbers or databases: B. subtilis BS1, 1945660; B. subtilis BS2, 2293156; M. thermoautotrophicum H, 1272331; M. thermoformicicum, 1279772; Myobacterium tuberculosis, 1722951; N. gonorrhoeae, University of Oklahoma Genome Center; N. meningitidis, Sanger Centre; Streptococcus pneumoniae, The Institute for Genomic Research; Streptococcus pyogenes, University of Oklahoma Genome Center. Identical amino acids are lightly shaded; darkly shaded amino acids in white are conserved among all  carbonic anhydrase sequences. Numbering refers to that of the Cab amino acid sequence.

Expression of Cab in M. thermoautotrophicum. We have previously shown that carbonic anhydrase activity (0.8 U/mg) is present in M. thermoautotrophicum cell extract (48). Western blot analysis was performed to determine if this activity is at least in part due to expression of Cab in M. thermoautotrophicum. In addition to an antiserum raised against Cab, antisera raised against the Anabaena strain PCC7120  carbonic anhydrase and M. thermophila  carbonic anhydrase (Cam) were used. A cross-reacting protein of the correct size for Cab was detected by Western blot analysis using the antiserum raised to Cab (Fig. 4). No proteins cross-reacting to the antiserum raised against the  carbonic anhydrase were detected; however, a protein cross-reacting to the antiserum raised against the  carbonic anhydrase from M. thermophila was detected (Fig. 4). Analysis of the genome sequence of M. thermoautotrophicum revealed an ORF encoding a protein with 30.7% identity to Cam; however, it is not yet known if the protein encoded by this ORF has carbonic anhydrase activity. Thus, the carbonic anhydrase activity detected in M. thermoautotrophicum may be due to the presence of both  and  carbonic anhydrases.

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Western blot analysis for carbonic anhydrase in cell extract protein from M. thermoautotrophicum. Each lane was loaded with 50 µg of M. thermoautotrophicum H cell extract protein and probed with either preimmune serum (lane 1) or antiserum to the Anabaena strain PCC7120  carbonic anhydrase (lane 2), M. thermoautotrophicum  carbonic anhydrase Cab (lane 3), or M. thermophila  carbonic anhydrase Cam (lane 4); detection was with anti-rabbit immunoglobulin G-alkaline phosphatase conjugate. Positions of molecular mass markers are indicated to the left in kilodaltons.

Conclusions. The results presented here are the first demonstration of plant-type (-class) carbonic anhydrases in the Archaea. The heterologously produced carbonic anhydrase from M. thermoautotrophicum, designated Cab, is the first documented  carbonic anhydrase from a thermophile and is thermostable at temperatures up to 75°C. It is anticipated that the thermophilic nature of Cab will facilitate the determination of a crystal structure, the first for any enzyme of the  class. The presence of  carbonic anhydrase in a thermophilic archaeon suggests that this enzyme may play a more widespread role in prokaryotic physiology than previously thought.