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Journal of Bacteriology, October 1999, p. 6247-6253, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Plant-Type (
-Class) Carbonic Anhydrase in the Thermophilic
Methanoarchaeon Methanobacterium
thermoautotrophicum
Kerry S.
Smith and
James G.
Ferry*
Department of Biochemistry and Molecular Biology, The
Pennsylvania State University, University Park, Pennsylvania 16802
Received 21 April 1999/Accepted 30 July 1999
 |
ABSTRACT |
Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of
carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of
highly evolved eukaryotes. Here we report on the first known plant-type
(
-class) carbonic anhydrase in the archaea. The
Methanobacterium thermoautotrophicum
H cab
gene was hyperexpressed in Escherichia coli, and the
heterologously produced protein was purified 13-fold to apparent
homogeneity. The enzyme, designated Cab, is thermostable at
temperatures up to 75°C. No esterase activity was detected with
p-phenylacetate as the substrate. The enzyme is an apparent
tetramer containing approximately one zinc per subunit, as determined
by plasma emission spectroscopy. Cab has a CO2 hydration
activity with a kcat of 1.7 × 104 s
1 and Km for
CO2 of 2.9 mM at pH 8.5 and 25°C. Western blot analysis indicates that Cab (
class) is expressed in M. thermoautotrophicum; moreover, a protein cross-reacting to
antiserum raised against the
carbonic anhydrase from
Methanosarcina thermophila was detected. These results show
that
-class carbonic anhydrases extend not only into the
Archaea domain but also into the thermophilic prokaryotes.
 |
INTRODUCTION |
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).
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MATERIALS AND METHODS |
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 M
1 cm
1). 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
(Mr = 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;
pKa 8.4)-28.6 mM Na2SO4-48.6 µM
m-cresol purple. The steady-state parameters
kcat and
kcat/Km 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 |
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).

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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.
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Most dicotyledonous plant carbonic anhydrases that have been purified
and characterized are reported to be dependent on a
reducing agent to
retain catalytic activity. The oxidized, inactive
pea enzyme could be
reactivated to only 60% of the original activity
by the addition of a
reducing agent (
26,
27). Purification
in the presence of
reducing agents or the addition of reducing
agents to purified Cab had
no affect on the catalytic activity
(
47). Thus, Cab joins
the other two prokaryotic

carbonic anhydrases
that are insensitive
to oxidation (
20,
49).
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.

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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).
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(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).
Although all three classes of carbonic anhydrase are inhibited by the
same types of compounds, inhibition constants vary considerably
between
individual enzymes. Detailed structural information for
enzyme-inhibitor complexes exists only for the

class carbonic
anhydrases, primarily human isozymes CA I and CA II (
37).
Anions
prevent formation of the coordinated hydroxide ion, which is
essential
in the catalytic hydration of CO
2 (
35,
36). Nitrate belongs
to a group of anions that appear to bind
close to the metal ion
without displacing the critical zinc-bound water
molecule necessary
for activity (
38). Conversely, another
group of anions including
chloride, iodide, and azide directly
coordinate to the metal ion
and displace the zinc-bound water molecule
(
32,
40). The
class carbonic anhydrases are only weakly
inhibited or not inhibited
by divalent anions such as sulfate
(
43); thus, sulfate is only
an inhibitor for plant enzymes
belonging to the
class.
Cab is also susceptible to inhibition by the sulfonamides acetazolamide
and ethoxyzolamide (IC
50s of 0.06 and 0.007 mM,
respectively).
Sulfonamides inhibit the activity of carbonic anhydrases
by binding
to the active site zinc via the nitrogen atom of the
sulfonamide
group (
37). Differences in the affinity of the
sulfonamides
have been explained by subtle differences in the active
sites
of the human isozymes; therefore, it is not surprising to see
differences between the different classes of carbonic
anhydrases.
(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.
According to quaternary structure, the

carbonic anhydrases are
divided into three groups represented by the dicotyledon,
monocotyledon, and nonplant enzymes (
7). The native
molecular
masses of the enzymes from dicotyledenous plants have been
reported
to vary between 140 and 250 kDa, with a subunit mass of 24 to
34 kDa. The oligomeric state has been recently shown to be an
octamer,
consisting of two covalently linked tetramers (
7).
Carbonic
anhydrases from monocotyledenous plants have been suggested
to be
dimers (
19). CynT from
E. coli has been reported
to be
either a tetramer or a dimer, depending on the experimental
conditions
(
20), and the

carbonic anhydrase identified
in the unicellular
green alga
Coccomyxa sp. has also been
shown to be a tetramer
(
23). Thus, the quaternary structure
of Cab is like that of
the enzymes from
E. coli and
Coccomyxa sp.
(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 Km value for
CO2 is similar to those of the characterized enzymes of the
class; however, the kcat 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 kcat and in the catalytic
efficiency (kcat/Km) 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.
The values determined for Cab are the first kinetic parameters reported
for a prokaryotic

carbonic anhydrase. Among the
prokaryotic
carbonic anhydrases that have been kinetically characterized,
the
N. gonorrhoeae 
-class enzyme (
13) has the
highest
kcat value and is as catalytically
efficient (
kcat/
Km) as
the high-activity
human isozyme, CA II (
29,
52). The
Km values for CO
2 for the

and

prokaryotic carbonic anhydrases (
1,
13) are over
fivefold greater than that of Cab, suggesting that Cab may have
a
physiological role distinct from those of the prokaryotic

-
and

-class
enzymes.
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.
An alignment of the sequences that form a clade with Cab is shown in
Fig.
3. Indicated in this alignment are
six amino acid
residues that are 100% conserved among the 62 sequences
of putative

carbonic anhydrases identified in the search. Extended
X-ray
absorption fine structure studies of the spinach enzyme suggest
that the active-site zinc is coordinated by two cysteine residues
and
one histidine residue (
10,
42). Cysteine-32, histidine-87,
and cysteine-90 of Cab are conserved among all of the sequences
and
would be expected to be the ligands for the active-site zinc
in Cab;
however, a structure is needed for definitive proof. Three
other
residues in Cab that are conserved among these sequences
are
asparagine-34, arginine-36, and glycine-50, suggesting an
important
structural or catalytic role. The alignment shown in
Fig.
3 reveals
additional residues that are 100% conserved among
the members of this
clade.

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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.
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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.

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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.
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Cab is the first documented carbonic anhydrase from a
CO
2-reducing chemolithoautotrophic methanoarchaeon. These
microbes have
a high demand for CO
2 in both catabolic and
anabolic reactions,
suggesting that carbonic anhydrase may be essential
to deliver
CO
2 to the cell and concentrate it in the
vicinity of CO
2-utilizing
enzymes. This is analogous to the
role of carbonic anhydrase in
photosynthetic organisms in which the
enzyme is essential for
efficient CO
2 transport into the
cell and elevation of the CO
2 concentration near the active
site of the CO
2-fixing enzyme ribulose
bisphosphate
carboxylase (
5). For example, Cab could convert
bicarbonate
to CO
2, the substrate for the formylmethanofuran
dehydrogenase
(
57) catalyzing the first committed step of
methanogenesis.
In addition to utilizing CO
2 in its energy
metabolism,
M. thermoautotrophicum is a chemolithoautotroph,
synthesizing cell carbon from CO
2. The
central anabolic
pathways for
M. thermoautotrophicum are the autotrophic
pathway for acetyl coenzyme A biosynthesis and the incomplete
reductive
tricarboxylic acid cycle (
44). Some of the CO
2
fixation
enzymes in these pathways utilize
HCO
3
; thus, interconversion between
CO
2 and HCO
3
is another potential
role for Cab. Similar mechanisms requiring
carbonic anhydrase may also
facilitate the growth of anaerobes
which obtain energy by reducing
carbon dioxide to either acetate
(
Acetobacterium woodii and
Clostridium thermoaceticum) or methane
(
Methanobacterium formicicum and
Methanospirillum
hungateii).
In fact, carbonic anhydrase activity has been detected
in these
anaerobes, and Western blot analysis has identified proteins
that
cross-react to antisera raised against Cab (
12,
48).
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.
 |
ACKNOWLEDGMENTS |
We thank John Coleman and Birgit Alber for the generous gifts of
antisera. We also thank Cheryl Ingram-Smith for critical reading of the manuscript.
The work was supported by NIH-GM44661 and NASA-Ames cooperative
agreement NCC-1057.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, 205 South Frear Laboratory,
The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-5721. Fax: (814) 863-6217. E-mail:
jgf3{at}psu.edu.
 |
REFERENCES |
| 1.
| Alber, B. E., C. M. Colangelo, J. Dong,
C. M. V. Stalhandske, T. T. Baird, C. Tu, C. A. Fierke, D. N. Silverman, R. A. Scott, and J. G. Ferry. Kinetic and spectroscopic characterization of the gamma
carbonic anhydrase from the methanoarchaeon Methanosarcina
thermophila. Biochemistry, in press.
|
| 2.
|
Alber, B. E., and J. G. Ferry.
1994.
A carbonic anhydrase from the archaeon Methanosarcina thermophila.
Proc. Natl. Acad. Sci. USA
91:6909-6913[Abstract/Free Full Text].
|
| 3.
|
Alber, B. E., and J. G. Ferry.
1996.
Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.
J. Bacteriol.
178:3270-3724[Abstract/Free Full Text].
|
| 4.
|
Armstrong, J. M.,
D. V. Myers,
J. A. Verpoote, and J. T. Edsall.
1966.
Purification and properties of human erythrocyte carbonic anhydrases.
J. Biol. Chem.
241:5137-5149[Abstract/Free Full Text].
|
| 5.
|
Badger, M. R., and G. D. Price.
1994.
The role of carbonic anhydrase in photosynthesis.
Annu. Rev. Plant Physiol. Plant Mol. Biol.
45:369-392.
|
| 6.
|
Bjorkbacka, H.,
I. M. Johansson, and C. Forsman.
1999.
Possible roles for His 208 in the active-site region of chloroplast carbonic anhydrase from Pisum sativum.
Arch. Biochem. Biophys.
361:17-24[Medline].
|
| 7.
|
Bjorkbacka, H.,
I. M. Johansson,
E. Skarfstad, and C. Forsman.
1997.
The sulfhydryl groups of Cys 269 and Cys 272 are critical for the oligomeric state of chloroplast carbonic anhydrase from Pisum sativum.
Biochemistry
36:4287-4294[Medline].
|
| 8.
|
Boone, D. R.,
W. B. Whitman, and P. Rouviere.
1993.
Diversity and taxonomy of methanogens, p. 35-80.
In
J. G. Ferry (ed.), Methanogenesis: ecology, physiology, biochemistry, and genetics. Chapman & Hall, London, England.
|
| 9.
|
Boriack-Sjodin, P. A.,
R. W. Heck,
P. J. Laipis,
D. N. Silverman, and D. W. Christianson.
1995.
Structure determination of murine mitochondrial carbonic anhydrase V at 2.45-Å resolution: implications for catalytic proton transfer and inhibitor design.
Proc. Natl. Acad. Sci. USA
92:10949-10953[Abstract/Free Full Text].
|
| 10.
|
Bracey, M. H.,
J. Christiansen,
P. Tovar,
S. P. Cramer, and S. G. Bartlett.
1994.
Spinach carbonic anhydrase: investigation of the zinc-binding ligands by site-directed mutagenesis, elemental analysis, and EXAFS.
Biochemistry
33:13126-13131[Medline].
|
| 11.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 12.
|
Braus-Stromeyer, S. A.,
G. Schnappauf,
G. H. Braus,
A. S. Gossner, and H. L. Drake.
1997.
Carbonic anhydrase in Acetobacterium woodii and other acetogenic bacteria.
J. Bacteriol.
179:7197-7200[Abstract/Free Full Text].
|
| 13.
|
Chirica, L. C.,
B. Elleby,
B. H. Jonsson, and S. Lindskog.
1997.
The complete sequence, expression in Escherichia coli, purification and some properties of carbonic anhydrase from Neisseria gonorrhoeae.
Eur. J. Biochem.
244:755-760[Medline].
|
| 14.
|
Eriksson, A. E., and A. Liljas.
1993.
Refined structure of bovine carbonic anhydrase-III at 2.0 angstrom resolution.
Proteins Struct. Funct. Genet.
16:29-42.
[Medline] |
| 15.
|
Ferry, J. G.
1999.
Enzymology of one-carbon metabolism in methanogenic pathways.
FEMS Microbiol. Rev.
23:13-38[Medline].
|
| 16.
|
Fukuzawa, H.,
S. Fujiwara,
A. Tachiki, and S. Miyachi.
1990.
Nucleotide sequences of two genes CAH1 and CAH2 which encode carbonic anhydrase polypeptides in Chlamydomonas reinhardtii.
Nucleic Acids Res.
18:6441-6442[Free Full Text].
|
| 17.
|
Fukuzawa, H.,
S. Fujiwara,
Y. Yamamoto,
M. L. Dionisio-Sese, and S. Miyachi.
1990.
cDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii regulation by environmental CO2 concentration.
Proc. Natl. Acad. Sci. USA
87:4383-4387[Abstract/Free Full Text].
|
| 18.
|
Gornall, A. G.,
C. J. Bardawill, and M. M. David.
1948.
Determination of serum proteins by means of the Biuret reaction.
J. Biol. Chem.
177:751-766.
|
| 19.
|
Graham, D.,
M. L. Reed,
B. D. Patterson,
D. G. Hockley, and M. R. Dwyer.
1984.
Chemical properties, distribution, and physiology of plant and algal carbonic anhydrases.
Annu. N. Y. Acad. Sci.
429:222-237[Medline].
|
| 20.
|
Guilloton, M. B.,
J. J. Korte,
A. F. Lamblin,
J. A. Fuchs, and P. M. Anderson.
1992.
Carbonic anhydrase in Escherichia coli. A product of the cyn operon.
J. Biol. Chem.
267:3731-3734[Abstract/Free Full Text].
|
| 21.
|
Hakansson, K.,
M. Carlsson,
L. A. Svensson, and A. Liljas.
1992.
Structure of native and apo carbonic anhydrase-II and structure of some of its anion-ligand complexes.
J. Mol. Biol.
227:1192-1204[Medline].
|
| 22.
|
Hewett-Emmett, D., and R. E. Tashian.
1996.
Functional diversity, conservation, and convergence in the evolution of the -, -, and -carbonic anhydrase gene families.
Mol. Phylogenet. Evol.
5:50-77[Medline].
|
| 23.
|
Hiltonen, T.,
H. Bjorkbacka,
C. Forsman,
A. K. Clarke, and G. Samuelsson.
1998.
Intracellular beta-carbonic anhydrase of the unicellular green alga Coccomyxa. Cloning of the cDNA and characterization of the functional enzyme overexpressed in Escherichia coli.
Plant Physiol.
117:1341-1349[Abstract/Free Full Text].
|
| 24.
|
Holmquist, B.
1988.
Elimination of adventitious metals.
Methods Enzymol.
158:6-12[Medline].
|
| 25.
|
Huang, S.,
Y. Xue,
E. Sauer-Eriksson,
L. Chirica,
S. Lindskog, and B. H. Jonsson.
1998.
Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide.
J. Mol. Biol.
28:301-310.
|
| 26.
|
Johansson, I. M., and C. Forsman.
1993.
Kinetic studies of pea carbonic anhydrase.
Eur. J. Biochem.
218:439-446[Medline].
|
| 27.
|
Johansson, I. M., and C. Forsman.
1994.
Solvent hydrogen isotope effects and anion inhibition of CO2 hydration catalysed by carbonic anhydrase from Pisum sativum.
Eur. J. Biochem.
224:901-907[Medline].
|
| 28.
|
Kannan, K. K.,
B. Notstrand,
K. Fridborg,
S. Lovgren,
A. Ohlsson, and M. Petef.
1975.
Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-Å resolution.
Proc. Natl. Acad. Sci. USA
72:51-55[Abstract/Free Full Text].
|
| 29.
|
Khalifah, R. G.
1971.
The carbon hydroxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isozymes B and C.
J. Biol. Chem.
246:2561-2573[Abstract/Free Full Text].
|
| 30.
|
Kisiel, W., and G. Graf.
1972.
Purification and characterization of carbonic anhydrase from Pisum sativum.
Phytochemistry
11:113-117.
|
| 31.
|
Kisker, C.,
H. Schindelin,
B. E. Alber,
J. G. Ferry, and D. C. Rees.
1996.
A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila.
EMBO J.
15:2323-2330[Medline].
|
| 32.
|
Kumar, V.,
K. K. Kannan, and P. Sathyamurthi.
1994.
Differences in anion inhibition of human carbonic anhydrase I revealed from the structures of iodide and gold cyanide complexes.
Acta Crystallogr.
D50:731-738.
|
| 33.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of the bacteriophage T7.
Nature
227:680-685[Medline].
|
| 34.
|
Larsson, S.,
H. Bjorkbacka,
C. Forsman,
G. Samuelsson, and O. Olsson.
1997.
Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula × tremuloides.
Plant Mol. Biol.
34:583-592[Medline].
|
| 35.
|
Lindskog, S.
1982.
Carbonic anhydrase, p. 115-170.
In
G. L. Eichorn, and L. G. Marzilli (ed.), Advances in inorganic chemistry, vol. 4. Elsevier/North Holland, Amsterdam, The Netherlands.
|
| 36.
|
Lindskog, S.
1983.
Carbonic anhydrase, p. 78-121.
In
T. G. Spiro (ed.), Zinc enzymes. John Wiley & Sons, New York, N.Y.
|
| 37.
|
Lindskog, S.
1997.
Structure and mechanism of carbonic anhydrase.
Pharmacol. Ther.
74:1-20[Medline].
|
| 38.
|
Mangani, S. H., and K. Hakansson.
1992.
Crystallographic studies of the binding of protonated and unprotonated inhibitors to carbonic anhydrase using hydrogen sulphide and nitrate anions.
Eur. J. Biochem.
210:867-871[Medline].
|
| 39.
|
Mori, K.,
Y. Ogawa,
K. Ebihara,
N. Tamura,
K. Tashiro,
T. Kuwahara,
M. Mukoyama,
A. Sugawara,
S. Ozaki,
I. Tanaka, and K. Nakao.
1999.
Isolation and characterization of CA XIV, a novel membrane-bound carbonic anhydrase from mouse kidney.
J. Biol. Chem.
274:15701-15705[Abstract/Free Full Text].
|
| 40.
|
Nair, S. K., and D. W. Christianson.
1993.
Crystallographic studies of azide binding to human carbonic anhydrase II.
Eur. J. Biochem.
213:507-515[Medline].
|
| 41.
|
Nolling, J.,
T. D. Pihl,
A. Vriesema, and J. N. Reeve.
1995.
Organization and growth phase-dependent transcription of methane genes in two regions of the Methanobacterium thermoautotrophicum genome.
J. Bacteriol.
177:2460-2468[Abstract/Free Full Text].
|
| 42.
|
Rowlett, R. S.,
M. R. Chance,
M. D. Wirt,
D. E. Sidelinger,
J. R. Royal,
M. Woodroffe,
Y. F. Wang,
R. P. Saha, and M. G. Lam.
1994.
Kinetic and structural characterization of spinach carbonic anhydrase.
Biochemistry
33:13967-13976[Medline].
|
| 43.
|
Simonsson, I., and S. Lindskog.
1982.
The interaction of sulfate with carbonic anhydrase.
Eur. J. Biochem.
123:29-36[Medline].
|
| 44.
|
Simpson, P. G., and W. B. Whitman.
1993.
Anabolic pathways in methanogens, p. 445-472.
In
J. G. Ferry (ed.), Methanogenesis: ecology, physiology, biochemistry, and genetics. Chapman & Hall, London, England.
|
| 45.
|
Sly, W. S., and P. Y. Hu.
1995.
Human carbonic anhydrases and carbonic anhydrase deficiencies.
Annu. Rev. Biochem.
64:375-401[Medline].
|
| 46.
|
Smith, D. R.,
L. A. Doucette-Stamm,
C. Deloughery,
H. Lee,
J. Dubois,
T. Aldredge,
R. Bashirzadeh,
D. Blakely,
R. Cook,
K. Gilbert,
D. Harrison,
L. Hoang,
P. Keagle,
W. Lumm,
B. Pothier,
D. Qiu,
R. Spadafora,
R. Vicaire,
Y. Wang,
J. Wierzbowski,
R. Gibson,
N. Jiwani,
A. Caruso,
D. Bush,
H. Safer,
D. Patwell,
S. Prabhakar,
S. McDougall,
G. Shimer,
A. Goyal,
S. Pietrokovski,
G. M. Church,
C. J. Daniels,
J.-I. Mao,
P. Rice,
J. Nolling, and J. N. Reeve.
1997.
Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics.
J. Bacteriol.
179:7135-7155[Abstract/Free Full Text].
|
| 47.
| Smith, K. S., and J. G. Ferry.
Unpublished data.
|
| 48.
| Smith, K. S., C. Jakubzick, T. S. Whittam, and
J. G. Ferry. Carbonic anhydrase is widespread in prokaryotes
and dates to the origin of life. Submitted for publication.
|
| 49.
|
So, A. K., and G. S. Espie.
1998.
Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803.
Plant Mol. Biol.
37:205-215[Medline].
|
| 50.
|
Soltes-Rak, E.,
M. E. Mulligan, and J. R. Coleman.
1997.
Identification and characterization of a gene encoding a vertebrate-type carbonic anhydrase in cyanobacteria.
J. Bacteriol.
179:769-774[Abstract/Free Full Text].
|
| 51.
|
Stams, T.,
S. K. Nair,
T. Okuyama,
A. Waheed,
W. S. Sly, and D. W. Christianson.
1996.
Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-Å resolution.
Proc. Natl. Acad. Sci. USA
93:13589-13594[Abstract/Free Full Text].
|
| 52.
|
Steiner, H.,
B. H. Jonsson, and S. Lindskog.
1976.
The catalytic mechanism of human carbonic anhydrase C: inhibition of CO2 hydration and ester hydrolysis by HCO3 .
FEBS Lett.
62:16-20[Medline].
|
| 53.
|
Stetter, K. O.
1996.
Hyperthermophiles in the history of life, p. 1-18.
In
G. R. Bock, and J. A. Goode (ed.), Evolution of hydrothermal ecosystems on Earth (and Mars?). John Wiley & Sons, Chichester, England.
|
| 54.
|
Studier, F. W., and B. A. Moffat.
1986.
Use of the bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
J. Mol. Biol.
189:113-130[Medline].
|
| 55.
|
Tabor, S., and C. C. Richardson.
1985.
A bacteriophage T7 RNA polymerase/promoter system for controlled expression of specific genes.
Proc. Natl. Acad. Sci. USA
82:1074-1078[Abstract/Free Full Text].
|
| 56.
|
Thompson, J. D.,
T. J. Gibson,
F. Plewniak,
F. Jeanmougin, and D. J. Higgins.
1997.
The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res.
25:4876-4882[Abstract/Free Full Text].
|
| 57.
|
Vorholt, J. A., and R. K. Thauer.
1997.
The active species of `CO2' utilized by formylmethanofuran dehydrogenase from methanogenic Archaea.
Eur. J. Biochem.
248:919-924[Medline].
|
| 58.
|
Wilbur, K. M., and N. G. Anderson.
1948.
Electrometric and colorimetric determination of carbonic anhydrase.
J. Biol. Chem.
176:147-154[Free Full Text].
|
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