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Plant Physiol. (1998) 117: 1341-1349
Intracellular
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Carbonic anhydrase (CA) (EC 4.2.1.1)
enzymes catalyze the reversible hydration of CO2, a
reaction that is important in many physiological processes. We have
cloned and sequenced a full-length cDNA encoding an intracellular
-CA from the unicellular green alga Coccomyxa.
Nucleotide sequence data show that the isolated cDNA contains an open
reading frame encoding a polypeptide of 227 amino acids. The predicted
polypeptide is similar to -type CAs from Escherichia
coli and higher plants, with an identity of 26% to 30%. The
Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically
characterized. The mature protein is a homotetramer with an estimated
molecular mass of 100 kD. The CO2-hydration activity of the
Coccomyxa enzyme is comparable with that of the pea
homolog. However, the activity of Coccomyxa CA is
largely insensitive to oxidative conditions, in contrast to similar
enzymes from most higher plants. Fractionation studies further showed
that Coccomyxa CA is extrachloroplastic.
CA (EC 4.2.1.1) is a zinc-containing enzyme that catalyzes the
reversible reaction CO2 + H2O Conversely, the CAs belonging to the Chloroplastic The kinetic characteristics of chloroplast The rate of the uncatalyzed interconversion between the two inorganic
carbon species, CO2 and
HCO3 Some species of green algae and cyanobacteria that are photosynthetic
components of lichens also possess CCMs, but there are some species
that appear to lack this mechanism. The green alga Coccomyxa
is one such photobiont (Palmqvist et al., 1994 The aim of this study was to characterize the major intracellular
Determination of Internal Amino Acid Sequences
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
HCO3
+ H+. CA is widely distributed throughout nature,
from eukaryotes such as vertebrates, invertebrates, and plants, to
prokaryotes such as archaeabacteria and eubacteria. The enzyme is
classified into three independent CA gene families designated 
, ,
and 
(Hewett-Emmett and Tashian, 1996
). The -CAs are found
primarily in animals (Tashian, 1992), but homologs have also been
identified in the bacterium Neisseria gonorrhoeae
(Hewett-Emmett and Tashian, 1996) and the green alga
Chlamydomonas reinhardtii (Fukuzawa et al., 1990). This is
the most extensively studied CA family, and includes the biochemically
well-characterized mammalian CA isozymes, the crystal structures of
which have been solved to high resolution (Kannan et al., 1975;
Eriksson et al., 1988a; Eriksson and Liljas, 1993; Boriack-Sjodin et
al., 1995).
-CAs are a newly discovered gene family, with the
enzyme from Methanosarcina thermophila being the only -CA isolated and characterized thus far (Alber and Ferry, 1994). Related sequences have been found in several eubacteria and in Arabidopsis (Hewett-Emmett and Tashian, 1996), but it is not known as yet whether
they encode functional CAs. The crystal structure for the -CA from
M. thermophila is different from that of the -type enzymes. The -CA is trimeric, with the active site situated between the subunits (Kisker et al., 1996).
-CA family have been found in both
C3 and C4 monocot and dicot
plants, in the mitochondria of C. reinhardtii, and in
various eubacteria (Eriksson et al., 1996; Hewett-Emmett and Tashian,
1996). Among the dicot species, the sequence similarity between the
different -CAs is around 80% (60% identity). The homology is
slightly lower between the monocot and dicot homologs, but the
similarity remains considerable (>70%). In contrast, the -CAs
found in prokaryotes are more variable and exhibit low sequence similarity to the plant homologs (30%). Alignment of all known amino
acid sequences from functional -CAs reveals invariant amino acid
residues at 26 positions, of which most are found within two regions.
Extended radiographic-absorption fine-structure analysis of spinach CA
suggests a Cys-His-Cys-H2O ligand scheme for
binding of the zinc ion (Bracey et al., 1994; Rowlett et al., 1994).
The first of these invariant Cys residues is found in one conserved region, whereas the His and the other Cys residue are situated in a
second conserved region. However, to our knowledge, no
three-dimensional structure has been described for any -CA. Most of
the biochemical studies have been done on chloroplastic homologs from
C3 dicots and on the Escherichia coli
enzyme.
-CA is nuclear encoded and synthesized in the
cytoplasm with an N-terminal transit peptide that targets the precursor
into the chloroplast stroma (Forsman and Pilon, 1995). Subsequent
maturation involves removal of the transit peptide, folding, and
oligomerization. The native molecular masses of CAs from
C3 dicot plants have been reported to vary
between 140 and 250 kD, with a subunit mass of 26 to 34 kD, each
binding one zinc ion (Reed and Graham, 1981). The -CA multimeric
complex has been shown to consist of eight subunits (Aliev et al.,
1986; Björkbacka et al., 1997). CAs from monocot plants have a
monomeric mass of around 25 kD and an estimated native mass of 42 kD
(Atkins et al., 1972; Atkins, 1974). The CA from E. coli is
also reported to be an oligomer, most likely a tetramer or a dimer,
depending on the experimental conditions (Guilloton et al., 1992).
-CA have been studied for
both pea (Johansson and Forsman, 1993) and spinach enzymes (Pocker and
Ng, 1973; Rowlett et al., 1994). Both possess a high catalytic
efficiency, with Kcat values between
105 and 106
s1 at high pH. The kinetic mechanism for these
enzymes is consistent with the general mechanism proposed for the
high-activity -CA isozymes (Silverman and Lindskog, 1988).
, is insufficient to cope
with the metabolic demand within a plant cell, but this varies among
different organisms (for review, see Badger and Price, 1994). Because
CA catalyzes the reversible hydration of CO2, it
has been proposed that chloroplastic CA is involved in the fixation of
CO2 in the Calvin cycle, which involves the carboxylating enzyme Rubisco (Reed and Graham, 1981).
CO2 is the only substrate for Rubisco, but
HCO3 is the predominant carbon
species within the alkaline chloroplast stroma (Makino et al., 1992).
In addition, CA appears to play a role in the CCM present in certain
cyanobacteria and free-living algae (for review, see Badger, 1987).
). The Coccomyxa genus is composed of many species forming
symbiotic relationships in a small but diverse group of lichens
(Honegger, 1991). This alga is unusual in that it lacks a CCM but has a
very high intracellular CA activity (Hiltonen et al., 1995). The
specific biological function of this internal CA in
Coccomyxa is unknown at present, as is its subcellular
location.
-CA from Coccomyxa. By cloning and sequencing the
corresponding cDNA, we were able to overexpress the enzyme in E. coli and purify it to homogeneity. Structural and kinetic studies
of this new -CA demonstrated that the Coccomyxa enzyme
possesses several interesting properties distinct from the higher-plant
-CAs. Furthermore, we demonstrated that the Coccomyxa CA
is extrachloroplastic and probably located to the cytosol.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
). The gel was stained with 0.5% Coomassie brilliant blue in 20% methanol and 0.5% acetic acid to visualize the
25-kD polypeptide. The band was excised and subjected to digestion with
modified trypsin (Promega) according to the method of Rosenfeld et al.
(1992). The collected peptides were subjected to amino acid sequence
analysis using a sequencing system (model 476A, Applied Biosystems).
RNA Isolation and cDNA Library Construction
Total RNA was isolated from 2-L Coccomyxa cultures grown according to the method of Hiltonen et al. (1995). Cells were
centrifuged at 1,100g for 10 min at 4°C and the pellet
(3.7 g) was resuspended in 10 mL of 50 mM Tris-HCl, pH 7.6, and 10 mM EDTA. Cells were lysed in a precooled French
pressure cell (Aminco, Silver Spring, MD) at 160 MPa and immediately
mixed with 40 mL of 4 M guanidinium isothiocyanate, 50 mM Tris-HCl, pH 7.6, 10 mM EDTA, and 2%
sarcosyl. The mixture was centrifuged at 4,000g for 5 min at
4°C. CsCl was added to the supernatant to a final concentration of
40% (w/v), and the supernatant was centrifuged for 10 min at
31,000g at 4°C. The resulting supernatant was laid on a
5.7 M CsCl cushion and centrifuged for 18 h at
150,000g at 20°C. The pellet was resuspended in 200 µL
of prewarmed (56°C) RNA-elution buffer (mRNA isolation kit,
Stratagene) containing 1% SDS. The RNA sample was phenol extracted
once and precipitated with ethanol, then the pellet was resuspended in
100 µL of elution buffer. Polyadenylated RNA was isolated from total
RNA using an mRNA isolation kit. A Coccomyxa cDNA library
was made from 5 µg of the purified poly(A+) RNA
using a cDNA-synthesis kit (ZAP Express, Stratagene).
Screening of the cDNA Library
About 1.5 × 105 plaque-forming units were spread among Escherichia coli XL1-Blue MRF (Stratagene) host cells on agar plates and analyzed according to standard procedures (Sambrook et al., 1989) using a 550-bp DNA probe corresponding to the
3
end of the Coccomyxa CA. The probe was generated by PCR
amplification from the Coccomyxa cDNA library using the
degenerate primer
5-CGGGAATTCGCIGGIGTIACIAA(T/C)(T/C)TITGGAT-3, corresponding to the internal peptide sequence TAGVTNLWI, and the nondegenerate primer T7 (22-mer, Stratagene).
PCR was performed in a thermal cycler (Perkin-Elmer) using 1 × 107 plaque-forming units of the cDNA library.
Hybridization to the radiolabeled probe was carried out at 65°C for
15 h in 4× SSPE (1× SSPE = 150 mM NaCl, 1 mM EDTA, and 15 mM sodium phosphate, pH 7.4),
5× Denhardt's solution (0.05% Ficoll 400, 0.05% PVP, and 0.05%
BSA), 0.5% SDS, and denatured salmon-sperm DNA (100 µg
mL1). After hybridization the filters were
washed at 65°C in 2× SSPE, 0.5% SDS followed by 1× SSPE, 0.1%
SDS. Positive plaques were identified and isolated according to the
instructions of the manufacturer (Stratagene).
DNA Sequencing
Selected clones were sequenced by the dideoxy chain-termination method using T3 and T7 primers (Stratagene) and the fmol DNA sequencing system (Promega). DNA sequences were analyzed using Genetics Computer Group (Madison, WI) software (Devereux et al., 1984).
Expression of Coccomyxa CA in E. coli and Protein Isolation
A 786-bp fragment containing the complete coding region for the Coccomyxa CA, including an extra 86-bp 3 untranslated region, was obtained by PCR amplification using the primers
5-GCGGAATTCATCGAGGGACGCATGTCAGCTAAAGACACTGCC-3 and
5-CTCCATCTAGAGTCACCTTGTAGGCA-3, which contain
cleavage sites for EcoRI and XbaI,
respectively. The PCR product was digested with EcoRI and
XbaI, and then ligated into the expression vector pMAL-c2
(New England Biolabs) just downstream of and in-frame with the
malE gene encoding the MBP. The resulting MBP/-CA was expressed in E. coli and purified using an amylose resin
(New England Biolabs). For enzyme production, the cells were grown in
Luria-Bertani broth containing 0.2% Glc and 50 µg
mL1 carbenicillin at 37°C to an
A600 of 0.6. Isopropylthio--galactoside was added to a final concentration of 1.5 mM and the
incubation was continued for another 2 h.
1.
After washing with eight column-volumes of column buffer, the fusion
protein was eluted with 10 mM maltose in the same buffer. The sample was concentrated using a Centriprep 30 unit (Amicon, Beverly, MA) and incubated for 10 h with factor Xa (Boehringer Mannheim) at a final concentration of 0.5%. The cleaved fusion protein
was desalted using a PD-10 column (Pharmacia) before being loaded on a Q-Sepharose FF (Pharmacia) ion-exchange column
equilibrated with 20 mM Tris-HCl, pH 8.0, and 1 mM EDTA. Coccomyxa CA was eluted with a stepwise
gradient of 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, and 1 mM EDTA. Fractions containing CA activity were pooled
and loaded for one more passage through the affinity column. The
isolated protein was concentrated using a Centricon 10 device (Amicon), and fractions containing CA activity throughout the purification steps
were analyzed by SDS-PAGE (Laemmli, 1970). The purified CA was
sequenced using a sequencing system (model 476A, Applied Biosystems) to
verify that the N terminus was correct after cleavage of the fusion
protein.
Preparation of Antiserum
The purified Coccomyxa CA, in PBS (10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl and 2.5 mM KCl), was mixed with Freund's complete adjuvant and injected into rabbits (Agrisera AB, Vännäs, Sweden). Every 14 d the immunized rabbits were injected with another 100 µg of CA mixed with incomplete adjuvant.Protein Concentration
Protein concentrations were determined either according to the method of Bradford (1976) or by determining the
A280 for the purified enzyme. The molar
extinction coefficient (
) for Coccomyxa CA was determined
as described by Gill and von Hippel (1989), giving a value of
280 = 37,500 ± 3,500 M1 cm1. All
stated enzyme concentrations are subunit concentrations.
Protein Structure Analyses
The native molecular mass of the purified Coccomyxa CA was estimated by gel-filtration chromatography performed on a Sephacryl S-300H column (Pharmacia) equilibrated with 20 mM Tris-HCl and 0.1 M NaCl, pH 7.4. Ovalbumin (45 kD), aldolase (158 kD), catalase (240 kD), and ferritin (450 kD) (Combithek, Boehringer Mannheim) were used as protein standards. Purified Coccomyxa CA and soluble cell proteins were analyzed by 9% polyacrylamide native-PAGE. The proteins were blotted onto a nitrocellulose filter for immunoreaction tests with antiserum directed against CA from Coccomyxa in conjunction with horseradish peroxidase-conjugated secondary antibodies and an enhanced-chemiluminescence detection system (Amersham).1 and the path length was 1 mm. For near-UV
analysis the protein concentration was 1.0 mg
mL1 and the path length was 4 mm. Spectra
recorded for pea CA were obtained using the same protocol except that
the far-UV region was scanned in a spectrodichrograph (model CD6,
Jobin-Yvon Instruments SA, Longjumeau, France) using a sample
concentration of 0.5 mg mL1 and a 0.5-mm path
length. The samples contained 10 mM potassium phosphate
buffer, pH 7.5. The observed ellipticities were converted to mean
residue ellipticities (
) on the basis of a molecular mass of 24.7 kD
and 227 amino acids for Coccomyxa CA, and 24.2 kD and 221 amino acids for pea CA.
CA Activity Measurements
During enzyme purification, fractionation, activation, and inhibition studies, CO2-hydration activity was assayed at 2°C using the colorimetric method of Rickli et al. (1964).
Initial rates of CO2 hydration were measured at
578 nm using a sequential stopped-flow spectrofluorimeter (model
DX-17MV, Applied Photophysics, Leatherhead, UK) at 25°C by the
changing-pH-indicator method (Khalifah, 1971; Steiner et al.,
1975). The buffer/indicator pair was Taps
(3-[{2-hydroxy-1,1-bis(hydroxymethyl)etyl}amino]-1-proparesulfonic acid)/m-cresol purple with 10 µM EDTA.
The initial rates, calculated by fitting data from the first part
of the trace to a first-order rate equation, were fitted by nonlinear
regression to the Michaelis-Menten equation using the GraFit program
(Erithacus Software Ltd., London, UK).
Modification of Free Cys Residues with DTNB
The free thiol content was estimated from the increase in A412 caused by formation of a 2-nitro-5-thiobenzoate anion caused by cleavage of DTNB upon reaction with a thiolate anion. A molar extinction coefficient of 14,150 M1 cm1 was
used in the calculations (Riddles et al., 1983). The reactions were
carried out in 0.1 M potassium phosphate buffer, pH 7.3, 1 mM EDTA, in a spectrophotometer (model 320, Perkin-Elmer).
Enzyme concentrations were 0.5 to 3 µM, and DTNB was
added in a molar excess of 1000. Reduced enzyme samples were obtained
by incubation with 10 mM DTT or 100 mM
2-mercaptoethanol for 1 h, followed by extensive dialysis against
degassed phosphate buffer under N2.
Subcellular Fractionation
A 10% to 80% linear Percoll gradient was generated by mixing 100% Percoll with 2× breaking medium (1× breaking medium: 35 mM Hepes-KOH, pH 7.7, 375 mM sorbitol, 10 mM EDTA, 1 mM MnCl2, and 5 mM MgCl2) in a 1:1 ratio and centrifuging the solution at 40,000g for 1 h. One liter of Coccomyxa culture (5 µg mL1
chlorophyll) was centrifuged at 1,500g for 10 min at 4°C
and resuspended in 20 mL of 1× breaking medium. Cells were disrupted for 30 s in a precooled Bead Beaker (Biospec Products,
Bartlesville, OK) filled with 0.5-mm-diameter glass beads and cell
suspension (1:1 [v/v]). The disrupted cells were carefully layered
onto the Percoll gradient and centrifuged at 3,000g for 20 min at 4°C. Four distinct fractions were taken, each characterized
with a light microscope.
, except that 4 mM DTT rather than GSH was included in the assay medium to
ensure full activation of the enzyme. PEPC was measured as a marker for
the cytosol (Gardeström and Edwards, 1983). For all
marker-enzyme-activity measurements, changes in
A340 resulting from NAD(P)H cleavage were
monitored on a spectrophotometer (model DU-8, Beckman). Protein samples
from the fractionation step were separated on a 15% SDS-PAGE gel and
blotted onto a nitrocellulose filter (MSI, Westboro, MA). Antiserum
directed against Coccomyxa CA was used, and the
antibody-antigen conjugate was detected using horseradish
peroxidase-linked secondary antibodies and enhanced chemiluminescence
(Amersham).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cloning and Sequencing of -CA cDNA
) was digested with trypsin and the resulting peptides were separated by HPLC. Five different internal amino acid sequences were determined (Fig. 1). One of the sequences, TAGVTNLW, was
used to design a degenerate 18-base primer. Along with a 22-base primer
specific for the T7 promoter, the degenerate
oligonucleotide was used to amplify a 550-bp fragment from the
Coccomyxa cDNA library. Subsequent sequencing of the
fragment confirmed that it was derived from a cDNA encoding part of a
-CA gene.
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-CA probe to screen the
Coccomyxa cDNA library, from which 15 positive clones were
obtained. Three of the longest cDNAs were completely sequenced in both
directions, and all three were identical except for different amounts
of truncation at the 5 end. The longest of the three selected clones
was 1137 bp, consisting of an open-reading frame of 681 bp, with 198 and 258 untranslated nucleotides in the noncoding 5 and 3 regions,
respectively. The cDNA library was rescreened with a probe
corresponding to the 5 end of the CA cDNA. This fragment starts at
amino acid position 23 and stops 570 bp downstream. The second
screening gave an additional set of seven positive clones. These
clones, together with five of the unsequenced clones from the first
screening, were sequenced from the 5 end and 500 bp downstream and
were all found to be identical to the full-length cDNA. The
5-untranslated region contained stop codons in all three reading
frames. No Met codon was found in this region upstream of the start Met
codon. The deduced protein of 227 amino acid residues has a predicted
molecular mass of 24.7 kD and compared with other -CAs (Fig. 1) is
about 50% similar (26%-30% identical) to higher-plant and
eubacterial homologs.
Overexpression of Coccomyxa CA in E. coli
To characterize the Coccomyxa CA in more detail, the corresponding cDNA was overexpressed in E. coli as a fusion to the C terminus of the MBP. After purification of the fusion protein, the Coccomyxa CA was removed by factor Xa digestion, and then isolated by ion-exchange chromatography. The purified protein migrated as a single discrete band with a molecular mass of 25 kD on a SDS-PAGE gel (Fig. 2A). The CA has an apparent native molecular mass of approximately 100 kD, as indicated by gel-filtration chromatography (Fig. 2B). Western-blot analysis was performed to determine whether the oligomeric state of the overexpressed protein matched that of the Coccomyxa endogenous enzyme. Under nondenaturing conditions the overexpressed protein separated as a single complex, and corresponded to a similar-sized band in the crude extract (data not shown). Thus, this algal enzyme seems to be homotetrameric.
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Structural Analysis of Coccomyxa CA
In the far-UV region (Fig. 3A) the CD spectrum of the Coccomyxa enzyme has an intense positive band at 194 nm and two slightly weaker negative bands at 208 and 218 nm. This band pattern suggests that Coccomyxa CA has a high-helix structure content (Johnson, 1990). The higher-plant
homolog from pea also seems to be dominated by -helix structures,
and the spectra for the two enzymes suggest a similar content of
various secondary structure elements (Fig. 3A). In the near-UV region
there are extensive differences: Coccomyxa CA has a
predominantly positive CD spectrum, whereas the spectrum for pea CA is
dominated by a negative band at around 280 nm (Fig. 3B).
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Kinetic Properties
The Coccomyxa CA was found to have a high catalytic activity. Kinetic parameters for the CO2-hydration reaction were determined using the stopped-flow technique. Values of Kcat = (3.8 ± 0.1) × 105 s1 and Km = 4.7 ± 0.3 mM were obtained in 50 mM
Taps buffer, pH 8.7, at 25°C. These are somewhat higher than
the values reported for the pea CA (Johansson and Forsman, 1993),
whereas the
Kcat:Km ratio
of (8.0 ± 0.5) × 107
M1 s1 is
almost identical to that reported for the higher-plant CA. The
differences are comparatively small and could reflect subtle differences in buffer and pH dependencies. Levels of inhibition of the
CO2-hydration activity of Coccomyxa CA
caused by specific inhibitors are presented in Table
I, together with
Ki values for the pea homolog. A relatively
large difference was observed between the algal and pea enzymes in
their sensitivity to the sulfonamide inhibitors. The binding affinity
of ethoxyzolamide is almost 30 times higher for the pea enzyme than for
the Coccomyxa CA, whereas the binding affinity of
acetazolamide is more than 10 times higher for the algal protein. The
inhibition by anions showed only minor variations between the two CAs.
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Oxidation/Reduction of Coccomyxa CA
-CAs localized to higher plant chloroplasts have been reported
to be sensitive to oxidation and, therefore, are dependent on a
reducing environment to retain catalytic activity (Tobin, 1970; Atkins
et al., 1972; Cybulsky et al., 1979; Johansson and Forsman, 1993).
Oxidized pea CA required a reducing agent for maximal activation of the
enzyme (Johansson and Forsman, 1993). In contrast, the activity of
Coccomyxa CA was found to be independent of the
presence of a reducing agent. The CO2-hydration activity of enzyme purified without reductant in the isolation buffers
was high, and it remained constant when the enzyme was incubated
for 10 min in 100 mM 2-mercaptoethanol or 1 mM
DTT (data not shown). Thus, the catalytic activity of
Coccomyxa CA is not significantly affected by the oxidation
state. Analysis of the amino acid sequence for Coccomyxa CA
(Fig. 1) shows the presence of nine Cys residues in each subunit. Of
these, only the two Cys residues thought to act as zinc ligands are
conserved.
Cell-Fractionation Studies
Microscopic studies of the fractions generated by separation in a Percoll gradient showed an enrichment of seemingly intact chloroplasts in fraction 3, although it also contained aggregated material, probably derived from thylakoid membranes. Fraction 1 contained soluble proteins, derived from both the cytosol and broken organelles; fraction 2 contained thylakoid membranes; and fraction 4, the lowest fraction, contained intact cells. Measurements of marker-enzyme activities supported the microscopic observations (Table II). Most of the activity of both NADP-GAPDH (95%) and PEPC (100%) were detected in fraction 1, whereas no enzyme activity was detected in fractions 2 and 4. Fraction 3 contained activity for the chloroplast marker enzyme NADP-GAPDH (5%) but no PEPC activity. This confirms the presence of intact chloroplasts in fraction 3. Of the four fractions, CA activity was found only in fraction 1. Western-blot analysis confirmed the CA activity measurements, with CA protein detected only in fraction 1 (Fig. 4). No protein corresponding to Coccomyxa CA was detected in fraction 3, the fraction containing intact chloroplasts.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have isolated and characterized a cDNA encoding a -CA from
the alga Coccomyxa. Enzymes from the -CA family share
some common features with members of the other two CA families, -CAs and -CAs. They are all zinc enzymes that catalyze the reversible dehydration of HCO3 to
CO2, and they are all sensitive to similar kinds
of chemical inhibitors, including sulfonamides and monovalent anions.
Nevertheless, the -, -, and -CAs clearly belong to three
distinct gene families according to sequence homology (Hewett-Emmett
and Tashian, 1996). On the same basis, -CAs can be further divided
into three subgroups: those originating from eubacteria, dicot plants,
and monocot plants (Hewett-Emmett and Tashian, 1996). Alignment of the
deduced amino acid sequence for the Coccomyxa CA with other
known -CAs, however, shows that this enzyme cannot readily be
classed with any of these subgroups.
-untranslated region upstream from the putative start Met
does not contain any additional Met codons before the stop codons in
any of the three frames. The 5 ends of 12 positive clones were also
analyzed and all sequences were found to be identical except for
different amounts of truncation, which strongly indicates that the
sequence was reliably identified. In summary, the majority of CA
activity in Coccomyxa is located in the cytosol, although
the presence of as-yet-unidentified chloroplastic or mitochondrial CAs
cannot be excluded.
suggested that this CA was chloroplast located and that it had a role
similar to that of CA in C3 plants. A cytosolic
CA could also facilitate the diffusion of inorganic carbon from the
inner surface of the plasmalemma to the chloroplast envelope (Badger and Price, 1994). Moreover, the absence of a CCM in
Coccomyxa has previously been correlated with the relatively
more efficient Rubisco of this alga than that of algae possessing a CCM
(Palmqvist et al., 1995). Palmqvist et al. (1995) also suggested that
there was an extracellular CA, but so far we have been unable to
measure any periplasmic CA activity from intact Coccomyxa
cells. Another possibility is that the CA in Coccomyxa may
not be directly involved in photosynthesis. As suggested by Fett and
Coleman (1994), cytosolic CA may instead be required to catalyze the
formation of HCO3, the
substrate for cytosolic PEPC, in a role similar to that suggested for
the CA localized in the mesophyll cells of C4
plants.
), suggesting that the existence of cytosolic -CA may be common
to both algae and higher plants.
)
and spinach (Rowlett et al., 1994). Similarly, we observed structural
features common to the different -CAs. The primary structures
contain a high degree of identity. The content of secondary structure
elements seems to be similar, because the general outline of the CD
spectra in the far-UV region is very similar, suggesting a domination
of -helix structure. This highlights one of the structural
differences between the -, -, and -CAs: the - and -CAs
are composed mainly of -sheet structures (Kannan et al., 1975;
Eriksson et al., 1988a; Eriksson and Liljas, 1993; Boriack-Sjodin et
al., 1995; Kisker et al., 1996). However, Coccomyxa CA also shows several distinct properties that imply certain differences between the algal and higher-plant -CAs.
; Björkbacka et
al., 1997). Furthermore, the near-UV CD spectra of CAs from
Coccomyxa and pea differ extensively; the CD bands in this
wavelength region arise mainly from immobilized aromatic side chains
located in an asymmetric environment (Strickland, 1974). This region is
generally assumed to be indicative of the tertiary structure of the
protein. However, because the Coccomyxa and pea enzymes
differ in Trp content (having five and two Trp residues, respectively),
and the enzymes apparently possess distinct quaternary structures, the
different shapes of the near-UV CD spectra do not necessarily imply
different overall folding of the individual subunits. Furthermore, we
have studied pea CA mutants that assemble into tetramers rather than
wild-type octamers, and these mutants have predominantly positive CD
spectra in the near-UV region, with shapes and intensities very similar
to those of Coccomyxa CA (Björkbacka et al., 1997).
; Price et
al., 1992), whereas the CAs in pea and other C3
dicots are dependent on a reducing environment to retain catalytic
activity. Of the nine Cys residues in the Coccomyxa CA, only
the two proposed zinc ligands are conserved. Under oxidizing
conditions, the Coccomyxa CA apparently forms a single
disulfide bond. Only four Cys residues were modified by DTNB in the
oxidized enzyme, whereas five to six Cys residues were modified in the
reduced Coccomyxa CA. This bond is probably not formed
within the active-site region. Therefore, the catalytic activity of the
Coccomyxa CA remains unchanged whether the enzyme is
oxidized or reduced.
). Assuming
that the sulfonamide coordination to the zinc is similar in the
-CAs, the weaker binding of the more hydrophobic (and the stronger
binding of the more hydrophilic) sulfonamide to the
Coccomyxa CA than to the pea enzyme may reflect differences
in the hydrophobicity of the surfaces near the active site. This could
be limited to differences in one or a few residues interacting with the
aromatic part of the inhibitor.
-CAs that have been catalytically investigated
are from the higher plants pea (Johansson and Forsman, 1993) and
spinach (Pocker and Ng, 1973; Rowlett et al., 1994). In general, the
enzymatic characteristics for these -CAs are consistent with the
zinc-hydroxide mechanism proposed for -CAs (Steiner et al., 1975),
and it seems likely that the Coccomyxa CA also follows the
same general mechanism. The work presented here will provide a
foundation for a more detailed characterization of the physiological
function of CA in Coccomyxa under different growth
conditions, especially in comparison with the CA homologs in algae
possessing a CCM.
| |
FOOTNOTES |
|---|
Received February 12, 1998;
accepted May 5, 1998.
The accession number for the Coccomyxa -CA cDNA
sequence reported in this article is U49976.
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ABBREVIATIONS |
|---|
Abbreviations:
CA, carbonic anhydrase.
CCM, inorganic
carbon-concentrating mechanism.
CD, circular dichroism.
DTNB, 5,5-dithiobis(2-nitrobenzoic azid).
MBP, maltose-binding
protein.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
PEPC, PEP carboxylase.
| |
ACKNOWLEDGMENT |
|---|
We thank Dr. Bo Ek (Department of Cell Research, Swedish University of Agricultural Sciences, Uppsala, Sweden) for skillful determination of the amino acid sequences.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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-Carbonic Anhydrase of the Unicellular Green
Alga Coccomyxa
Vo)/(Vt 
