physiological and molecular biological characterization of ... · physiological and molecular...

Physiological and Molecular Biological Characterization ofIntracellular Carbonic Anhydrase from the Marine DiatomPhaeodactylum tricornutum1

Dan Satoh2, Yasutaka Hiraoka2, Brian Colman3, and Yusuke Matsuda*

Department of Chemistry, Kwansei-Gakuin University, 1–1–155 Uegahara, Nishinomiya 662–8501, Japan

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO2-grown cellsof the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growthCO2 conditions. Ethoxyzolamide (0.4 mm), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at lowconcentrations of dissolved inorganic carbon, whereas 2 mm acetazolamide had little effect on the affinity for dissolvedinorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism inP. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, andtwo-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. Thepurified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reducedand nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNAends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence withthe N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologousto �-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acidresidues that may constitute a CO2-binding site appeared to be unique among the �-CAs so far reported.

The aquatic environment is generally CO2 limitingfor photoautotrophs mainly because of the limitedcapacity of water to hold gaseous CO2 and the slowdiffusion rate of CO2(aq), which is about 10�4 timesthat in the atmosphere. It is well established that anumber of algae and cyanobacteria actively take upand accumulate dissolved inorganic carbon (DIC)intracellularly, which allows algal cells to photosyn-thesize efficiently even under atmospheric CO2. ThisDIC acquisition mechanism is termed a carbon con-centrating mechanism (CCM) and a number of work-ers have suggested that carbonic anhydrase (CA)plays a key role in the CCM (Kaplan and Reinhold,1999).

CA (EC 4.2.1.1) is a zinc-containing enzyme thatcatalyzes the reversible dehydration of HCO3

� toCO2. This reaction is known to play important rolesin various biological processes such as ion exchange,respiration, pH homeostasis, CO2 acquisition, andphotosynthesis (Tashian, 1989; Badger and Price,1994; Smith and Ferry, 2000, Moroney et al., 2001).

CAs are widely distributed in living organisms andare categorized, on the basis of their amino acidsequence, into three distinct families designated as�-, �-, and �-type (Hewett-Emmett and Tashian,1996; Smith et al., 1999; Smith and Ferry, 2000), whichdo not share epitopes and are thought to haveevolved independently (Smith et al., 1999). �-TypeCA occurs in animals, eubacteria, green algae, andcyanobacteria (Smith et al., 1999) and is typified bymammalian forms and two periplasmic forms in thegreen alga Chlamydomonas reinhardtii (Hewett-Emmett and Tashian, 1996; Smith and Ferry, 2000).�-Type CAs have been found in archaebacteria andeubacteria (Smith et al., 1999) and one has been pu-rified from the methanogen Methanosarcina thermo-philia (Alber and Ferry, 1994). A region of a gene forthe cyanobacterial carboxysomal protein, ccmM isalso known to be homologous to �-type CAs (Price etal., 1993; Smith et al., 1999). Both �- and �-type CAshave been shown to ligate with Zn by three Hisresidues (Kisker et al., 1996).

�-Type CAs occur ubiquitously and have been sep-arated into six phylogenetic clades (Smith and Ferry,1999; Smith et al., 1999). �-CAs from monocots anddicots constitute independent groups. Another twoclades are exclusively prokaryotic and the other twoare composed of mixture of sequences from the eu-carya to procarya domains, which include �-CAsfrom the green algae C. reinhardtii, and Coccomyxa sp.,the red alga Porphyridium purpureum, and the cya-nobacterium Synechocystis PCC 6803 (Smith et al.,1999). A high consensus sequence among �-CAs, that

1 This work was supported in part by a grant from Invitation forResearch Institute of Innovative Technology for the Earth ResearchProposals and in part by Kwansei-Gakuin University (SpecialGrant for Individual Researcher to Y.M. and a visiting professor-ship to B.C.).

2 These authors contributed equally to the paper.3 Present address: Department of Biology, York University, 4700

Keele Street, Toronto, Canada M3J 1P3.* Corresponding author; e-mail [email protected]; fax

81–798 –51– 0914.

Plant Physiology, August 2001, Vol. 126, pp. 1459–1470, www.plantphysiol.org © 2001 American Society of Plant Biologists 1459 www.plantphysiol.orgon April 22, 2019 - Published by Downloaded from Copyright © 2001 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

is Cys-Xaan-His-Xaa2-Cys, is known to constitute theZn coordination site (Hewett-Emmett and Tashian,1996). X-ray crystallographic studies clarified the ac-tive site of �-CAs from pea (Pisum sativum; Kimberand Pai, 2000), the red algae P. purpureum (Mitsu-hashi et al., 2000), Escherichia coli (Cronk et al., 2000),and Methanobacterium thermoautotrophicum (Strop etal., 2001). P. purpureum CA is thought to use anadditional amino acid to constitute the Zn coordina-tion site; Asp at two amino acids down toward theC-terminal end from the first Zn ligand, Cys, is alsoconsidered to ligate with Zn.

The intracellular location of CA seems to be a cru-cial factor for the operation of the CCM. One form ofcyanobacterial �-CA is present in the carboxysome.Cells transformed to express human CAII in the cy-toplasm resulted in a high CO2-requiring phenotypepresumably due to stimulation of HCO3

� dehydra-tion in the cytoplasm and therefore an efflux of CO2from the cells (Price and Badger, 1989). Inactivationof �-CA gene (icfA) was also found to induce a high-CO2-requiring phenotype (Fukuzawa et al., 1992). Ineukaryotic algae, chloroplastic CAs are thought to berequired to supply CO2 to Rubisco in the stromaand/or the pyrenoid where the predominant speciesof inorganic carbon is HCO3

� because of the alkalineconditions of the stroma (Badger and Price, 1994). Asa mechanism to enhance the rate of CO2 supply toRubisco in C. reinhardtii, it has been suggested that�-type CA (Cah3) is associated with photosystem IIat the lumenal side of the thylakoid membrane andcatalyzes the dehydration of HCO3

� in the lumen togive an ample efflux of CO2 to the stroma or thepyrenoid (Karlsson et al., 1998; Park et al., 1999,Moroney et al., 2001). These data clearly indicate thatintracellular CA plays a key role in the CCM when itfunctions in particular cellular locations.

Diatoms are widespread in aquatic environments,and marine species are considered to be some of themost important CO2 fixers in the hydrosphere (Apt etal., 1996). Two strains of Phaeodactylum tricornutumhave been documented to possess a CCM in that theycan take up both CO2 and HCO3

� actively (Colmanand Rotatore, 1995; Johnston and Raven, 1996). John-McKay and Colman (1997) reported that all P. tricor-nutum strains they studied possessed internal CA butthat some of them also possessed external CA. Exter-nal CA in P. tricornutum was also shown to be essen-tial for carbon acquisition under carbon-limited con-ditions (Iglesias-Rodriguez and Merrett, 1997).External CA therefore appears to operate to maintainequilibrium CO2 concentration in the periplasmiclayer from HCO3

�, predominant species of DIC inseawater. In contrast, the function of the internalform of CA in marine algae has not been studiedextensively. Only one diatom CA has been isolated,that from the marine diatom Thalassiosira weissflogii(TWCA1; Roberts et al., 1997; Cox et al., 2000), andthe structure of its Zn coordination site was deter-

mined by x-ray absorption spectrometry (Roberts etal., 1997; Cox et al., 2000). TWCA1 was shown toshare no homology with other CAs but Zn coordina-tion was made with three His ligands, which is astructure very similar to that of mammalian �-CAs(Roberts et al., 1997; Cox et al., 2000), suggesting aunique event of convergent molecular evolution.However, it is not clear whether or not the distinctstructure of TWCA1 is common in the CAs of otherdiatom species.

Detailed molecular studies of CA from more ma-rine diatom species is certainly needed because it isone of the critical enzymes for carbon acquisition inmarine microalgae. In this study, internal CA of themarine diatom P. tricornutum was related to the op-eration of the CCM physiologically and characterizedmolecular biologically.

RESULTS

Effects of Sulfonamide on PhotosyntheticAffinity for DIC

The effect of two specific inhibitors of CA,ethoxyzolamide (EZA) and acetazolamide (AZA),was used to determine the function of P. tricornutumCA in acquiring high-affinity photosynthesis (TableI). EZA is highly permeable to biological membranesand will inhibit both internal and external CA,whereas AZA is only weakly permeable and inhibitsonly external CA. EZA of 0.4 mm completely abol-ished high-affinity photosynthesis for DIC and DICconcentration at one-half-maximum rate of photo-synthesis (K0.5[DIC]) was 454 �m (Table I), whereaseither 0.4 or 2 mm AZA did not (Table I, K0.5[DIC]value of 29 and 44 �m, respectively). K0.5[DIC] valuewithout the addition of sulfonamide was found to be27 �m (Table I). Although the maximum rate of pho-tosynthesis (Pmax) was suppressed both by EZA treat-ment by about 39% and by 0.4 and 2 mm AZA treat-ment to 65% to 70% that of the control (Table I),photosynthetic rates at limited DIC was much lowerin EZA-treated cells than those in AZA-treated cells(data not shown). The absolute values of O2 evolu-tion rate at 100 �m of DIC in the medium were 20.0,18.0, 14.9, and 2.2 nmol mL�1 min�1 with controlcells, cells treated with 0.4 and 2 mm AZA, and 0.4mm EZA, respectively.

Table I. Photosynthetic parametersa determined in air-grown P.tricornutum in the presence and the absence of sulfonamides atpH 8.2 and 25°C

Treatment K0.5[DIC] Pmax

�M �mol O2 mg�1 Chl h�1

Control 27 � 1 209 � 152 mM AZA 44 � 9 147 � 240.4 mM AZA 29 � 6 135 � 300.4 mM EZA 454 � 87 81 � 16a Values are the mean � SD of three separate experiments.

Satoh et al.

1460 Plant Physiol. Vol. 126, 2001 www.plantphysiol.orgon April 22, 2019 - Published by Downloaded from Copyright © 2001 American Society of Plant Biologists. All rights reserved.


Measurement of CA Activity in Intact Cell or CellLysate of P. tricornutum

No CA activity was detected in intact cells of P.tricornutum grown in either air or high CO2 (Table II).However, CA activity was detected in cell lysates:26 � 9.5 Wilbur-Anderson (WA) units mg�1 Chlwere detected in high-CO2-grown cells, whereas114 � 38.7 WA units mg�1 Chl were found in air-grown cells (Table II). In a similar manner, strong CAactivity was detected on cellulose acetate plates afterelectrophoresis of extracts from air-grown cells,whereas those from high-CO2-grown cells containedvery weak activity at the same mobility on the plateas that from air-grown cells (Fig. 1). There were noisoforms detected on cellulose acetate plates (Fig. 1).

Purification of CA

The purification procedure of P. tricornutum CA issummarized in Table III. By ammonium sulfate pre-cipitation, about 42% of the activity of soluble CAwas retrieved. The specific activity of CA at this stagewas found to be 59 WA units mg�1 protein. Anion-exchange chromatography on a DEAE-sephacel col-umn increased the specific activity up to 3-fold andCA was eluted at about 110 mm Na2SO4 (data notshown). The fraction with CA activity was thensubjected to affinity chromatography on a p-amino-methylbenzene sulfone amide (p-AMBS) agarose col-umn and was eluted as a single peak by 25 mmTris-H2SO4 (pH 8.5) containing 0.3 m NaClO4 (Fig.2A). This step improved the specific activity about7.6-fold and the specific activity of CA was found tobe 1,114 WA units mg�1 protein. CA was purified to36.7-fold from the homogenate (Table III).

The purified CA gave a single band on SDS-PAGEunder both reducing and nonreducing conditions,confirming uniformity of this protein (Fig. 2B). Treat-ment with the reducing agent did not cause any shiftin the mobility of the band and the molecular massestimated by SDS-PAGE was 28 kD (Fig. 2B). AZAconcentration at one-half-maximum inhibition(I50[AZA]) of P. tricornutum CA was found to be 5 �10�8 M.

Molecular Cloning of Full-Length cDNA andDetermination of Nucleotide Sequence

The protein sequence of 50 amino acid residuesfrom the N terminus of the purified CA revealed that

this CA may be homologous to �-type CAs, whichprompted us to clone the CA gene of P. tricornutumwith the aid of the published sequences of �-typeCAs (Mitsuhashi and Miyachi, 1996; Hiltonen et al.,1998). The first PCR gave a mixture of hetero-sizedproducts ranging from 50 to 2,000 bp (data notshown). The second PCR gave several sizes of prod-ucts but the major one corresponded in size to ap-proximately 250 bp (data not shown). This 250-bpfragment was cloned into the plasmid vector and wassequenced. The deduced amino acid sequence of the250-bp fragment included a part of the N-terminalamino acid sequence of purified P. tricornutum CA(data not shown).

Utilizing the 250-bp fragment as a “core” sequence,the full-length cDNA sequence was determined by aRACE method with high-fidelity DNA polymerase(Fig. 3A). The full-length cDNA of P. tricornutum CAwas 995 bp and was shown to comprise 61 bp of a5�-untranslated region, 846 bp of an open readingframe, a termination codon, 58 bp of a 3�-

Table II. CA activitya of whole cells and supernatant of whole cellextracts of air- and high-CO2-grown P. tricornutum

Cells Whole Cells Supernatant of Extract

Air grown N.D.b 114 � 39High CO2 grown N.D. 26 � 9.5a (Tc/T � 1) mg�1 Chl. Values are the mean � SD of five separate

experiments. b N.D., Not detected.

Figure 1. CA activity after cellulose acetate electrophoresis of cell-free extracts of P. tricornutum grown in 5% (w/v) CO2 and air. CAactivity was visualized by soaking the plate in the pH-indicating dye,bromocresol purple, and exposing the plate to gaseous CO2.

Intracellular Carbonic Anhydrase of a Marine Diatom

Plant Physiol. Vol. 126, 2001 1461 www.plantphysiol.orgon April 22, 2019 - Published by Downloaded from Copyright © 2001 American Society of Plant Biologists. All rights reserved.


untranslated region, and 27 bp of poly(A�) tail (Fig.3A). The open reading frame (Fig. 3A, 62–907) en-codes a polypeptide of 282 amino acids (Fig. 3A,�46–236), whereas the N-terminal amino acid se-quence of purified CA started at Ala 46 amino acidsdown toward C terminus from the initial Met (Fig.3A). These data indicate that the mature CA in P.tricornutum can be categorized as a �-type and iscomposed of 236 amino acids (Fig. 3A, �1–236)whose molecular mass was calculated to be 26,354 D.The 46 amino acids at the upstream of N terminus ofmature CA appear to be a signal sequence (Fig. 3B).

RNA and Genomic DNA Gel-Blot Analysis

The size of CA mRNA was found to be 1.0 kb byRNA gel-blot analysis (Fig. 4A), which agreed wellwith the size of CA cDNA (995 bp). The same hy-bridization signal was clearly detected for transcriptsfrom high-CO2-grown cells, but its intensity was sig-nificantly lower than that of air-grown cells (Fig. 4A).The number of CA genes in the genome was esti-mated by Southern-blot analysis (Fig. 4B). CA cDNAdid not possess any recognition sites for any of thefour restriction enzymes used. The 5�-RACE productof CA cDNA (1–574) was prepared as a probe. One ortwo hybridization bands with different mobilitieswere detected in the four digests; one band wasobserved in BamHI, EcoRV, and PstI digest, whereastwo bands were observed in EcoRI digest (Fig. 4B),suggesting the presence of at least one intron thatcontains an EcoRI site.

Comparison of Deduced Amino Acid Sequence ofP. tricornutum CA with CAs in Other Species

The sequence similarity analysis of P. tricornutumCA was carried out with the DNA Data Bank ofJapan homology search system. Eight sequences thatwere moderately homologous to P. tricornutum CAwere retrieved, namely CA in Coccomyxa sp.(Hiltonen et al., 1998), N and C halves of CA1 inP. purpureum (Mitsuhashi and Miyachi, 1996), CynTproduct in E. coli (Guilloton et al., 1992), and CAs inpea (Majeau and Coleman, 1991), Spinacea oleracea(Burnell et al., 1989), M. thermoautotrophicum (Smithand Ferry, 1999), and Saccharomyces cerevisiae. Theidentities of the amino acid were 25.9% (Coccomyxasp.), 20.0% (P. purpureum), 26.2% (E. coli), 20.1% (pea),25.5% (S. oleracea), 20.9% (M. thermoautotrophicum),and 22.3% (S. cerevisiae), respectively (Fig. 5). Thesesequences are aligned at their maximum match inFigure 5 and amino acid residues that have beenshown either to constitute Zn ligands or to orienttoward Zn (Kimber and Pai, 2000; Mitsuhashi et al.,2000) are indicated by circles and asterisks, respec-tively. Given the alignment, Zn-binding residueswere found to be highly conserved, whereas aminoacids possibly related to the catalytic site (Kimberand Pai, 2000; Mitsuhashi et al., 2000) appeared to bedifferent in P. tricornutum CA (Fig. 5). The aminoacid, Leu70, does not correspond to any other known�-CAs and Phe92 does not correspond to �-CAs ofeither algal or higher plant origin but it correspondsto that in S. cerevisiae (Fig. 5).

Figure 2. Purification of P. tricornutum CA on ap-AMBS agarose column (A) and subsequentSDS-PAGE analysis (B). A, The column (2 mL ofbed volume) was pre-equilibrated with 50 mM

Bicine-NaOH (pH 8.3) and washed with 25 mM

Tris-H2SO4 containing 0.1 M Na2SO4. CA waseluted with 25 mM Tris-H2SO4 containing 0.3 M

NaClO4 with the flow rate at 0.2 mL min�1. B,Molecular mass markers (lane M) and 10 �g ofprotein from the peak fraction (no. 46) was ap-plied to 12% (w/v) polyacrylamide gel. CA wastreated with 0.5% (w/v) SDS in the presence(lane R) and absence (lane NR) of 5% (v/v)�-mercaptoethanol prior to the electrophoresis.

Table III. Summary of purification procedure of CA from P. tricornutum

Fraction Total Protein Total CA Activity Specific Activity Purification

mg WAUa WAUa mg�1 protein fold

Homogenate 197.9 6,175 31 1.0(NH4)2SO4 44.4 2,617 59 1.9DEAE-sephacel 8.9 1,444 163 5.2p-AMBS-agarose 0.37 423 1,144 36.7

a WA unit; Tc/T � 1.

Satoh et al.



DISCUSSION

The marine environment differs from that of fresh-water primarily in salinity and alkalinity, which cre-ates distinct DIC equilibria in seawater (Goyet andPoisson, 1989) and stable pH at slightly above 8.0.The DIC equilibrium at this pH gives rise to a lowCO2 concentration that may cause CO2 limitation tophotoautotrophs. However, it has been shown inmost of the marine microalgae so far investigatedthat under light-saturating conditions, photosynthe-sis is saturated at air equilibrium CO2 concentrations,presumably due to an efficient use of HCO3

�, whichis abundant in seawater (Raven, 1997; Tortell et al.,1997). The occurrence of direct HCO3

� uptake has

been shown in various marine microalgae (Burns andBeardall, 1987; Colman and Rotatore, 1995; Nimer etal., 1997, Raven, 1997). The data obtained in thepresent study also showed that photosynthetic O2evolution rate at limited [DIC] greatly exceeded theactual CO2 formation rate in the bulk medium,strongly suggesting a direct uptake of HCO3

�; irre-spective of the presence of AZA, O2 evolution rate at100 �m DIC was about 15 to 20 nmol mL�1 min�1,which is about 30-fold the un-catalyzed rate of CO2formation (Matsuda et al., 2001) in artificial seawaterenriched with one-half-strength of Guillard’s “F” so-lution (f/2; F2AW; Harrison et al., 1980) at pH 8.2. AnHCO3

�-dependent photosynthesis was also observed

Figure 3. Sequences of cDNA and deducedamino acids of P. tricornutum CA (A) and acomparison of N-terminal sequence with thoseof some algal proteins (B). A, The amino acidsequence is numbered from the N terminus ofthe mature protein. The putative cleavage site ofsignal peptide is indicated by a white triangle.The single-underlined region was initially se-quenced at protein bases upon purification ofP. tricornutum CA. Double underline indicatesan overlapped sequence between the 5�-RACEand the 3�-RACE products. Arrows indicateprimers used for the RACE. The terminationcodon and the putative polyadenylation signalare indicated by an asterisk and a box, respec-tively. B, Forty-six amino acids of the N terminusof P. tricornutum CA (Pha CA) were comparedwith fucoxanthin chlorophyll protein 3 ofP. tricornutum (Pha FCP3), internal CA of Chlor-ella sorokiniana (Csoro CA), periplasmic CA,CAH1, of C. reinhardtii (Chlam CAH1), andplasma membrane CA of Dunaliella salina(Duna CA). Hydrophobic amino acid residuesare indicated by white letters on a gray back-ground. The white triangles indicate putativecleavage sites of signal peptides.




in another strain of P. tricornutum (UTEX640; Mat-suda et al., 2001).

As an alternative mechanism for HCO3� use from

the bulk medium, CA, located in the periplasmicspace, has been shown to facilitate an indirect use ofHCO3

� by catalyzing CO2 formation close to theplasmalemma. This external CA has been shown tooccur both in marine algae (including P. tricornutum)and in freshwater algae (Aizawa and Miyachi, 1986;Williams and Colman, 1993; Iglesias-Rodriguez et al.,1997; Nimer et al., 1999). However, recent studieshave shown that external CA is not necessarily es-sential for growth under CO2 limitation in C. rein-hardtii (Moroney and Somanchi, 1999; Van and Spal-ding 1999) but rather that internal CA activity may becrucial under these conditions (Funke et al., 1997).P. tricornutum UTEX642 was previously shown tolack external CA (John-Mckay and Colman, 1997). Inthe present study, no external CA activity was de-tected either in air- or high-CO2-grown cells of P.tricornutum. This was confirmed further by sulfon-amide inhibition experiments in which up to 2 mmAZA did not lower the photosynthetic affinity forDIC (Table I). A single major band of CA activity,detected on cellulose acetate plates after electro-phoresis of lysates of both air- and high-CO2-growncells (Fig. 1), thus appeared to be an intracellularform of CA, which is encoded by a single gene,

and no other isoform was detected (Fig. 1) in thisstudy. However, these data do not exclude the pos-sibility of the occurrence of trace levels of other CAisoforms that may also have a role in the CCM in P.tricornutum.

The function of internal CA in the CCM of marinemicroalgae is largely unknown. The data in thepresent study demonstrate that EZA drastically in-hibited high-affinity photosynthesis repeat and con-firm the results of previous studies on P. tricornutum(Badger et al., 1998). Because EZA is also believed topossess direct inhibitory effects on photosystem IIand DIC uptake (Badger et al., 1998), the reason forthe EZA inhibition of high-photosynthetic affinity forDIC is not clear at present. Nevertheless, it has beenshown in most of the freshwater algae studied thatCA plays a crucial role in the operation of the CCMand this may hold true also in the case of marinediatoms. In the present study, EZA reduced Pmax toabout 40% that of the control and a similar, butweaker effect was also observed by prolonged treat-ment with high concentrations of AZA, presumablydue to permeation of AZA at trace concentration intothe cells. Therefore, it is plausible that internal CAmay also take part in supplying CO2 to photosynthe-sis and determine the Pmax in P. tricornutum. EZAtreatment of the green alga Chlorella ellipsoidea alsolowered the cells’ affinity for DIC to the level thatresembled that of high-CO2-grown cells, but Pmaxwas not affected by EZA (Y. Matsuda, unpublisheddata).

Internal CA detected in this study is clearly a low-CO2-inducible protein and this regulation occurs atthe transcriptional level even though there was aconstitutive level of accumulation of transcript ob-served in high-CO2-grown cells. The photosyntheticaffinity of P. tricornutum cells for DIC appeared toincrease concomitant with internal CA activity (datanot shown). However, it has been indicated that theregulation of CA expression in response to CO2 is notnecessarily related to the CCM. The thylakoid-lumenal form of CA, Cah3, in C. reinhardtii appearedto be expressed semiconstitutively despite changes inthe ambient [CO2], whereas the expression of theperiplasmic CAs, Cah1 and Cah2, and the mitochon-drial CAs, �-CA1 and �-CA2, which are possibly lesscrucial isoforms with respect to the operation of theCCM, have been shown to be highly dependent onchanges in the ambient [CO2] (Moroney and Chen,1998). The regulation mechanism of internal CA ex-pression in response to CO2 is yet to be studied andto be related functionally with CCM in P. tricornutum.

P. tricornutum CA was purified effectively byanion-exchange chromatography on DEAE-sephaceland on p-AMBS, an affinity column with a sulfon-amide ligand. CA from P. tricornutum was elutedfrom the p-AMBS column with 0.3 m NaClO4 (Fig. 1),which is a lower concentration than that used for the�-type CAs in C. sorokiniana (Satoh et al., 1998) and C.

Figure 4. RNA (A) and DNA (B) gel-blot analyses of cloned CA. A,Total RNA (15 �g) obtained from P. tricornutum grown in 5% (v/v)CO2 and air was separated on 1.0% (w/v) agarose gel, blotted ontonylon membrane, and hybridized with the 5�-RACE product of CAlabeled by alkaline-phosphatase. Hybridization signal was visualizedby fluorography using CDPstar (top half). Quantities of RNAs werenormalized with rRNA (bottom half). The molecular size correspondsto 1.0 kb and is indicated by a white arrow. B, Ten micrograms ofgenomic DNA was digested independently with four restriction en-zymes, BamHI, EcoRI, EcoRV, and PstI, and was separated by 0.8%(w/v) agarose gel, blotted onto nylon membrane, and hybridized withthe CA probe described above. Hybridization signal was visualizedas described above. The molecular sizes are indicated by whitearrows.

Satoh et al.



reinhardtii (Yang et al., 1985; Rawat and Moroney,1991). It should be noted that the affinity of P. tricor-nutum CA for sulfonamides might be lower thanthose in known �-CAs but higher than that of �-CAisolated from microalgae, i.e. AZA concentration atone-half-maximum inhibition (I50[AZA]) of P. tricor-nutum. CA was found to be about seven and 17 timeshigher than those observed in �-CA from C. sorokini-ana (Satoh et al., 1998) and C. reinhardtii CAH1 and 2(Tachiki et al., 1992), respectively, but 20 times lowerthan that of Coccomyxa �-CA (Hiltonen et al., 1995).

The primary structure of P. tricornutum CA showsthat it can be classified as a �-type CA and is homol-ogous to �-CAs in other algae and higher plants(Figs. 3A and 5). The molecular mass of P. tricornu-tum CA was estimated to be 28 kD by SDS-PAGE(Fig. 2B), whereas it is calculated to be 26.4 kD fromthe primary structure of the mature protein (Fig. 3A).This difference in molecular mass of approximately 2

kD may indicate the possible occurrence of posttrans-lational modification such as glycosylation, but apotential attachment site for an N-linked carbohy-drate chain (Asn-Xaa-Ser/Thr) was not found in thepolypeptide sequence.

Although the subcellular location of P. tricornutumCA is not known, this enzyme is soluble and there-fore seems to be located in the hydrophilic matrix ofthe cell and not in the membranes. This is in contrastto the internal CAs in C. reinhardtii of which about95% of the total CA is insoluble (Funke et al., 1997)and one isoform, Cah3, was postulated to function inthe CCM as a thylakoid membrane-associated form(Raven, 1997; Karlsson et al., 1998; Park et al., 1999).In contrast, the predominant form of internal CA inChlorella spp. was shown to be soluble when the cellshave an operational CCM (Williams and Colman,1993; Satoh et al., 1998). The amino acid sequence ofP. tricornutum CA may give some indication of its

Figure 5. Comparison of sequences of �-CAs from several different origins. �-CA of P. tricornutum (Pha) was compared withthat of Coccomyxa sp. (Coc; accession no. U49976), N and C half of P. purpureum �-CA (PorN and PorC, respectively;accession no. D86050), E. coli Cyn T product (Eco; accession no. M23219), chloroplastic �-CA of pea, (Pis; accession no.M63627), chloroplastic �-CA of S. oleracea (Spi; accession no. M27295), �-CA of S. cerevisiae (Sac; accession no. Z71312),and �-CA of M. thermoautotrophicum (Met; accession no. AE000918). The row of Pha is numbered from the N terminusamino acid sequence of the putative �-CA precursor deduced from the full-length cDNA. Sequences were aligned withCLUSTAL W at their maximum match and the amino acids identical with those of P. tricornutum CA are shown as whiteletters on black background. Circles and asterisks indicate putative zinc-ligand residues and residues orient toward Zn,respectively. White and black symbols are marked to the alignment based upon the information from the x-ray crystallo-graphic analyses of P. purpureum CA and pea CA, respectively.




intracellular location. The N-terminal region, fromthe first Met to Ser (Fig. 3A, �46 to �1) shows thetypical features of a signal sequence (von Heijne,1983) which includes several hydrophobic core resi-dues in the N termini,: the residues at the position �1and �3 are small, and the position at �2 is bulky.This region has similarities to the signal peptide ofthe fucoxanthin-chlorophyll a/c proteins (FCPs) inP. tricornutum (Grossman et al., 1990), C. reinhardtiiCAH1 (Fukuzawa et al., 1990), C. sorokiniana CA(Satoh et al., 1998), and the plasma membrane CA inD. salina (Fisher et al., 1996; Fig. 3B). This region wasalso predicted to be a signal for import to the endo-plasmic reticulum by analysis with TargetP, which isa neural network algorithms (Emanuelsson et al.,2000) and several other analyses for predictingprotein-targeting sites. It has been shown in P. tricor-nutum that three proteins in the fucoxanthin-chlorophyll a/c complex associated with photosystemII are encoded by the nuclear genome and have theendoplasmic reticulum signal peptide (Grossman etal., 1990). Bhaya and Grossman (1991) further sug-gested that some of the cytoplasmically synthesizedproteins of P. tricornutum required an endoplasmicreticulum signal to be directed to the plastids becausethe signal peptide of FCP3, one of the FCPs, can becotranslationally processed and transported into mi-crosomes in vitro. This suggests that P. tricornutumCA could be located in organelles. The intracellularlocation of P. tricornutum CA needs to be determinedcytochemically and physiologically to investigate itsbiochemical and cell structural organization whenCA functions in relation to carbon acquisition inmarine autotrophs.

The tertiary structures of �-type CAs have beendetermined by x-ray crystallography using enzymesobtained from the unicellular red algae, P. purpureum(Mitsuhashi et al., 2000), the pea plant (Kimber andPai, 2000), and M. thermoautotrophicum (Strop et al.,2001). It is interesting that despite the striking simi-larity in the primary structures of the active site, theirtertiary structures and proposed mechanisms of ca-talysis were quite different among these �-CAs. Thesubunit of P. purpureum CA was shown to be dividedinto two almost identical parts, i.e. N and C halvesthat revealed the typical primary structure of �-CA(Mitsuhashi et al., 2000). Each one-half contained aZn coordination site constituted of four amino acidresidues, Cys, Asp, His, and Cys, which forms anactive center with a hydrophobic domain primarilycomposed of Pro, Phe, and Leu in the other one-halfchain (Mitsuhashi et al., 2000). Binding of Zn by fouramino acid ligands, including that of Asp, and acooperative conformation to utilize trans-locatedsubdomains are a unique feature of the active centerof CA and hence the CO2 hydration mechanism isthought to use water, which does not bind to Zndirectly (Mitsuhashi et al., 2000). In contrast to thisunique structure of red algal �-CA, pea CA was

shown to possess an active site that is a mirror imageof that of �-CA and thus is assumed to share a similarmechanism of catalysis to �-CAs (Kimber and Pai,2000).

The tertiary structure of P. purpureum �-CA wasshown to be unique as described above and hencemight not occur in P. tricornutum �-CA (Fig. 5). If thecatalysis mechanism of P. tricornutum CA resemblesthat of �-CA as in the case of pea CA, the criticalamino acid residues involved in the CO2 hydrationmay be quite different from that of pea. The residuesHis42, Leu70, and Phe92, which correspond toGln151, Phe179, and Tyr205 in pea CA, may form aCO2-binding site, and the proton of the imidazolegroup in His42 would interact with the oxygen ofCO2 in place of the amide group in Gln151 in pea CA(Kimber and Pai, 2000).

The subunit structure of �-CAs varies dependingon species. Higher plant �-CA possess hexa- to oc-tameric structures (Sultemeyer et al., 1993; Kimberand Pai, 2000) whereas those in Monera and Protistahave been shown to possess di- to tetrameric struc-tures (Hiltonen et al., 1998; Mitsuhashi et al., 2000).The molecular mass of the native form of P. tricornu-tum CA could not be determined by gel filtrationbecause no distinct sharp peak was detected and theCA was distributed in a broad range of fractions thatcorrespond to the molecular mass of 800 to 29 kD(data not shown). This result was always obtainedwith different batches of preparations (data notshown), suggesting that the quaternary structure ofthis enzyme may be very large and be disassembledduring purification or by interaction with the matrixof the column. In an alternate manner, monomericpolypeptides might merely aggregate to form a largepseudo complex in vitro.

Intracellular CA appears to play a major role inacquiring and supplying substrate for photosynthesisin marine diatoms. The P. tricornutum CA describedin this study is the first �-CA to be detected in adiatom species. The other diatom CA so far isolatedis from T. weissflogii (TWCA1), which showed nohomology to the known algal CAs and therefore afundamental difference of carbon acquisition amongalgal phyla was proposed by the authors (Roberts etal., 1997). In contrast, the present study indicates thatthe primary structure of CA in a diatom is not mark-edly distinctive as far as one of the major neriticspecies, P. tricornutum, is concerned.

MATERIALS AND METHODS

Cells and Culture Condition

The marine diatom Phaeodactylum tricornutum (UTEX642) was obtained from the University of Texas CultureCollection (Austin) and was cultured axenically in F2AW(Harrison et al., 1980) under continuous illumination ofphoton flux density of 100 �mol m�2 s�1 at 20°C. The

Satoh et al.



culture was aerated with 5% CO2 (high-CO2-grown cells)or air (air-grown cells).

Determination of Photosynthetic Affinity for DIC

Cells were harvested by centrifugation at 1,500g for 10min at 25°C, washed twice with 350 �m NaCl buffered with10 mm Tris-HCl (pH6.8), and resuspended in CO2-freeF2AW buffered with 10 mm Tris-HCl (pH 8.2) under N2 ata chlorophyll a concentration of 10 �g mL�1. The rate ofphotosynthesis was measured with a Clark-type oxygenelectrode as described previously (Matsuda and Colman,1995) with or without the addition of CA inhibitors, EZA(0.4 mm; Sigma-Aldrich Japan, Tokyo) or AZA (0.4 or 2mm; Sigma-Aldrich Japan). The apparent K0.5[DIC] wasdetermined as described by Rotatore and Colman (1991).Cell suspension (1.5 mL) was placed in the O2 electrodechamber, illuminated with a fiber optic illuminator(Megalight100, Hoya-Schott Co., Tokyo) at a photon fluxdensity of 2,600 �mol m�2 s�1 and the cells allowed toreach CO2 compensation concentration. The photon fluxdensity was then increased to 6,400 �mol m�2 s�1 andaliquots of KHCO3 were added sequentially to the cellsuspension to create increasing DIC concentrations. Chlo-rophyll a concentration was determined by the spectropho-tometric method described by Jeffrey and Humphrey(1975).

Measurement of CA Activity

CA activity was measured by the potentiometric methoddescribed by Wilbur and Anderson (1948) with some mod-ifications. Twenty microliters of enzyme solution or cellsuspension was added to 1.48 mL of 20 mm Veronal buffer(pH 8.3) in a water-jacketed acrylic chamber maintained at4°C. The reaction was initiated by the addition of 0.5 mL ofice-cold CO2-saturated water and the time required for thepH to drop from 8.3 to 8.0 was determined. The WA unitwas defined as follows: WA unit � Tc/T � 1, where Tc andT are the time required for the pH drop in the absence andpresence of enzyme solution, respectively. For the qualita-tive determination of CA activity, electrophoresis on cellu-lose acetate plates (Titan III Zip Zone, Helena Laboratories,Mississauga, Canada) was carried out as described by Wil-liams and Colman (1993).

Extraction and Purification of CA

A culture of air-grown cells (20 L) at mid-logarithmicphase (OD730 0.2–0.4) was harvested by centrifugation at1,500g for 10 min at 25°C. The cells were resuspended in aminimum volume of 50 mm Bicine (N, N-Bis2-hydroxymethyl Gly)-NaOH buffer (pH 8.5) and thenmixed with 400 g of glass beads (0.105–0.125 mm: 0.177–0.250 mm: 0.350–0.500 mm � 1: 1: 2, Iuchi Seieido, Osaka).Cells were homogenized by vigorous vortexing for 1 minand chilled on ice for 5 min. This procedure was repeated15 times. A cell-free lysate was obtained from the homog-enate by centrifugation at 26,000g for 60 min at 4°C. CA

activity was recovered from the resulting supernatant byammonium sulfate precipitation between 30% and 65%saturation. This CA fraction was subjected to a two-steppurification using a DEAE-sephacel (Amersham PharmaciaBiotech, Little Chalfont, UK) column (i.d. 1.0 � 6.3 cm)followed by affinity chromatography on a p-AMBS agarose(Sigma Chemical Co., St. Louis) column (i.d. 0.8 � 4 cm).The DEAE-Sephacel column was equilibrated with 50 mmBicine-NaOH (pH 7.9) and protein was eluted with a lineargradient of Na2SO4 from 0 to 0.25 m. p-AMBS column wasequilibrated by 50 mm Bicine-NaOH (pH 8.3) and proteinwas eluted with 25 mm Tris-H2SO4 buffer containing 0.1 mNa2SO4 and the same buffer containing 0.3 m NaClO4.Protein concentration was determined by the Bradfordmethod using bovine serum albumin as the standard.

Determination of Molecular Mass

The CA fraction eluted from the p-AMBS agarose col-umn was applied to SDS-PAGE under both nonreducingand reducing conditions with 5% (v/v) �-mercaptoethanolusing a 12% (w/v) polyacrylamide gel and molecular-massstandards (DAIICHI III, Daiichi Pure Chemicals, Co., Ltd.,Tokyo). The molecular mass of CA was also determined bymolecular sieving on a Superdex 200 (Amersham Pharma-cia Biotech) column (i.d. 1.0 � 30 cm).

Determination of Amino Acid Sequence ofN-Terminal End

CA was blotted electrophoretically onto polyvinylidenedifluoride membrane (Immobilon, Millipore, Bedford, MA)followed by Coomassie Brilliant Blue staining. The CAband was cut out of the membrane and subjected to aminoacid sequencing analysis of the N-terminal end usingPPSQ-23 (Shimadzu Corp., Kyoto).

Extraction of Total RNA and MolecularCloning of CA cDNA

Air-grown cells (3.0 L) at late logarithmic phase(OD730 � 0.4) were harvested as described above. The cellswere frozen immediately, disrupted in liquid N2, and totalRNA was extracted as described by Chomczynski andSacchi (1987). cDNA was synthesized with M-MuLV Re-verse Transcriptase (New England Bio Labs Inc., Beverly,MA) and oligo (dT)15 primer.

Degenerate primers were designed according to theamino acid sequence of the N-terminal end of the purifiedCA (PtCAF1: 5�-CGC GGA TCC GAY ATI ACI GAR ATHTTY GAY GG-3�) and according to two published nucleo-tide sequences of �-CAs from Coccomyxa sp. (Hiltonen etal., 1998) and P. purpreum (Mitsuhashi and Miyachi, 1996;PtCAR1: 5�-CCG GAA TTC CCA TNG CNK CYT GIACHA T-3�), which corresponded to the sequences of DITE-IFDG and IVQ(A/D) AW(A/D), respectively. A partialnucleotide sequence of CA cDNA was amplified by PCR,on the assumption that P. tricornutum CA is homologous to�-CAs. The PCR profile was as follows: five cycles of 94°C




for 30 s, 50°C for 30 s, and 72°C for 90 s, followed by 35cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s. Theresulting PCR products were separated by agarose gelelectrophoresis and the bands corresponding in size toapproximately 400 to 600 bp were cut out of the gel and theextract was used as a template for a second PCR. Thenested-degenerate primers were designed according to theamino acid sequence of the N-terminal end of purified CA(PtCAF2: 5�-CCG GAA TTC GAY GCI GCN TAY TTY GAYAC-3�) and according to the two published nucleotide se-quences of �-CAs described above (PtCAR2: 5�-CCG GAATTC TAR TGI CCR CAN ACI ARD ATR TG-3�), whichcorresponded to the sequences of DAAYFDT and HIL-VCGH, respectively. The PCR profile was as follows: threecycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 90 s,followed by 37 cycles of 94°C for 30 s, 55°C for 30 s, and72°C for 90 s. The amplified fragment was purified byelectrophoresis and cloned into a plasmid vector (pT7BlueT-Vector, Novagen, Madison, WI). Cloned cDNA was se-quenced by using Thermo Sequenase Cycle sequence Kitand Cy5.5 labeled primers by the Long-Read Tower System(Amersham Pharmacia Biotech).

A full-length sequence of CA cDNA was determined byRACE method for the 5� and 3� ends of CA cDNA. SMARTRACE cDNA Amplification Kit and high-fidelity DNApolymerase (CLONTECH Laboratories Inc., Palo Alto, CA)were used according to the manufacturer’s protocols. CAgene-specific primers, PtCASP1: 5�-GGA TGC GTC GATGCC CGT GCT CCT CCG-3�, were designed based uponthe partial nucleotide sequence of CA cDNA. The 3�-RACEwas carried out with PtCASP1 and the universal primerprovided by manufacturer. The resulted PCR product wascloned into the plasmid vector and sequenced.

CA gene-specific primers, PtCASP2: 5�-GGT GAC ACGGGG ATC GCA GCA GGG GCG-3� and PtCASP3: 5�-GGAGGG GCA TGG TCC ACG TTG GCG ACG G-3�, weredesigned according to the cDNA sequence of 3� regiondetermined by 3�-RACE. The 5�-RACE was carried outwith PtCASP2 and the universal primer provided by man-ufacturer. The second PCR was carried out with PtCASP3and the universal primer provided by manufacturer asdescribed above on the product of first PCR and the re-sulted PCR product was cloned into a plasmid vector andsequenced. The sequence data was analyzed by the GENE-TYX System (Software development Co., Ltd., Tokyo). Ahomology search was carried out using the DNA DataBank of Japan homology search system (release 42, July2000) and the deduced amino acid sequence of P. tricornu-tum CA was compared with several known CA sequences(Burnell et al., 1989; Majeau and Coleman, 1991; Guillotonet al., 1992; Mitsuhashi and Miyachi, 1996; Hiltonen et al.,1998; Smith and Ferry, 1999).

RNA Gel-Blot Analysis

Total RNA (15 �g) from 5% (v/v) CO2- and air-growncells at mid-logarithmic phase was separated by 1.1% (v/v)formaldehyde-agarose gel electrophoresis and blotted ontonylon membrane (Hybond N�, Amersham Pharmacia Bio-

tech). The membrane was hybridized with the PCR productof 5�-RACE described above. The labeling of the probe andhybridization were carried out using Gene Images randomprime labeling module (Amersham Pharmacia Biotech).Hybridized probe was reacted with CDPstar (AmershamPharmacia Biotech) and visualized by fluorography.

Genomic DNA Gel-Blot Analysis

Two liters of high-CO2-grown cells (OD730 � 0.625) washarvested, frozen, and disrupted in liquid N2. The homog-enate was resuspended in 10 mm Tris-HCl (pH 8.0), 0.1 mEDTA, and 0.5% (w/v) sarcosyl and was treated with 40 �gmL�1 RNaseA (Sigma-Aldrich) at 37°C for 2 h followed bytreatment with 80 �g mL�1 proteinase K (Sigma-Aldrich)at 50°C for 2 h. Protein was removed with dialysis methodas described by Sambrook et al. (1989). Twelve microgramsof genomic DNA was digested separately with BamHI(Roche Diagnostics, Basel), EcoRI, EcoRV (Takara ShuzoCo., Ltd., Otsu, Japan), or PstI (Toyobo Co., Ltd., Osaka)and subjected to DNA gel-blot analysis. Probe preparationand visualization of hybridized band were carried out asdescribed in RNA gel-blot analysis.

ACKNOWLEDGMENT

The authors would like to thank Mr. Goro Hirozumi forhis technical assistance.

Received February 16, 2001; returned for revision April 26,2001; accepted May 18, 2001.

LITERATURE CITED

Aizawa K, Miyachi S (1986) Carbonic anhydrase and CO2

concentrating mechanisms in microalgae and cyanobac-teria. FEMS Microbiol Rev 39: 215–233

Alber BE, Ferry JG (1994) A carbonic anhydrase from thearchaeon Methanosarcina thermophila. Proc Natl Acad SciUSA 91: 6909–6913

Apt KE, Kroth-Pancic PG, Grossman AR (1996) Stablenuclear transformation of the diatom Phaeodactylum tri-cornutum. Mol Gen Genet 252: 572–579

Badger MR, Andrews TJ, Whitney SM, Ludwig M, Yel-lowlees DC, Leggat W, Price GD (1998) The diversityand coevolution of Rubisco, plastids, pyrenoids, andchloroplast-based CO2-concentrating mechanisms in al-gae. Can J Bot 76: 1052–1071

Badger MR, Price GD (1994) The role of carbonic anhy-drase in photosynthesis. Annu Rev Plant Physiol PlantMol Biol 45: 369–392

Bhaya D, Grossman A (1991) Targeting proteins to diatomplastids involves transport through an endoplasmic re-ticulum. Mol Gen Genet 229: 400–404

Burnell JN, Gibbs MJ, Mason JG (1989) Spinach chloro-plastic carbonic anhydrase: nucleotide sequence analysisof cDNA. Plant Physiol 92: 37–40

Burns BD, Beardall J (1987) Utilization of inorganic carbonby microalgae. J Exp Mar Biol Ecol 107: 1–21

Satoh et al.



Chomczynski P, Sacchi N (1987) Single-step method ofRNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 162: 156–159

Colman B, Rotatore C (1995) Photosynthetic inorganic car-bon uptake and accumulation in two marine diatoms.Plant Cell Environ 18: 919–924

Cox EH, McLendon GL, Morel FMM, Lane TW, PrinceRC, Pickering IJ, George GN (2000) The active site struc-ture of Thalassiosira weissflogii carbonic anhydrase 1. Bio-chem 39: 12128–12130

Cronk JD, O’Neill JW, Cronk MR, Endrizzi JA, ZhangKYJ (2000) Cloning, crystallization and preliminary char-acterization of a �-carbonic anhydrase from Escherichiacoli. Acta Crystal D Biol Crystallogr 56: 1176–1179

Emanuelsson O, Nielsen H, Brunak S, von Heijne G(2000) Predicting subcellular localization of proteinsbased on their N- terminal amino acid sequence. J MolBiol 300: 1005–1016

Fisher M, Gokhman I, Pick U, Zamir A (1996) A salt-resistant plasma membrane carbonic anhydrase is in-duced by salt in Dunaliella salina. J Biol Chem 271:17718–17723

Fukuzawa H, Fujiwara S, Yamamoto Y, Dionisio-SeseML, Miyachi S (1990) cDNA cloning, sequence, andexpression of carbonic anhydrase in Chlamydomonas rein-hardtii: regulation by environmental CO2 concentration.Proc Natl Acad Sci USA 87: 4383–4387

Fukuzawa H, Suzuki E, Komukai Y, Miyachi S (1992) Agene homologous to chloroplast carbonic anhydrase(icfA) is essential to photosynthetic carbon dioxide fixa-tion by Synechococcus PCC7942. Proc Natl Acad Sci USA89: 4437–4441

Funke RP, Kovar JL, Weeks DP (1997) Intracellular car-bonic anhydrase is essential to photosynthesis in Chlamy-domonas reinhardtii at atmospheric levels of CO2. PlantPhysiol 114: 237–244

Goyet C, Poisson A (1989) New determination of carbonicacid dissociation constants in seawater as a function oftemperature and salinity. Deep Sea Res 36: 1635–1654

Grossman A, Manodori A, Snyder D (1990) Light-harvesting proteins of diatoms: their relationship to thechlorophyll a/b binding proteins of higher plants andtheir mode of transport into plastids. Mol Gen Genet 224:91–100

Guilloton MB, Korte JJ, Lamblin AF, Fuchs JA, AndersonPM (1992) Carbonic anhydrase in Escherichia coli: a prod-uct of the cyn operon. J Biol Chem 267: 3731–3734

Harrison PJ, Waters RE, Taylor FJR (1980) A broad spec-trum artificial seawater medium for coastal and openocean phytoplankton. J Phycol 16: 28–35

Hewett-Emmett D, Tashian RE (1996) Functional diver-sity, conservation, and convergence in the evolution ofthe �-, �-, and �-carbonic anhydrase gene families. MolPhylogenet Evol 5: 50–77

Hiltonen T, Bjorkbacka H, Forsman C, Clarke AK, Sam-uelsson G (1998) Intracellular �-carbonic anhydrase ofthe unicellular green alga Coccomyxa: cloning of thecDNA and characterization of the functional enzymeoverexpressed in Escherichia coli. Plant Physiol 117:1341–1349

Hiltonen T, Karlsson J, Palmqvist K, Clarke AK, Sam-uelsson G (1995) Purification and characterization of anintracellular carbonic anhydrase from the unicellulargreen alga Coccomyxa. Planta 195: 345–351

Iglesias-Rodriguez MD, Merrett MJ (1997) Dissolved in-organic carbon utilization and the development of extra-cellular carbonic anhydrase by the marine diatom Phae-odactylum tricornutum. New Phytol 135: 163–168

Jeffrey SW, Humphrey GF (1975) New spectrophotometricequations for determining chlorophylls a,b,c1, and c2 inhigher plants, algae and natural phytoplancton. BiochemPhysiol Pflanzen 167: 191–194

John-McKay ME, Colman B (1997) Variation in the occur-rence of external carbonic anhydrase among strains ofthe marine diatom Phaeodactylum tricornutum (Bacillari-ophyceae). J Phycol 33: 988–990

Johnston AM, Raven JA (1996) Inorganic carbon accumu-lation by the marine diatom Phaeodactylum tricornutum.Eur J Phycol 31: 285–290

Kaplan A, Reinhold L (1999) CO2 concentrating mecha-nisms in photosynthetic microorganisms. Annu RevPlant Physiol Plant Mol Biol 50: 539–570

Karlsson J, Clarke AK, Chen ZY, Hugghins SY, Park YI,Husic HD, Moroney JV, Samuelsson G (1998) A novel�-type carbonic anhydrase associated with the thylakoidmembrane in Chlamydomonas reinhardtii is required forgrowth at ambient CO2. EMBO J 17: 1208–1216

Kimber MS, Pai EF (2000) The active site architecture ofPisum sativum �-carbonic anhydrase is a mirror image ofthat of �-carbonic anhydrases. EMBO J 19: 1407–1418

Kisker C, Schindelin H, Alber BE, Ferry JG, Rees DC(1996) A left-handed �-helix revealed by the crystalstructure of a carbonic anhydrase from the archaeonMethanosarcina thermophila. EMBO J 15: 2323–2330

Majeau N, Coleman JR (1991) Isolation and characteriza-tion of a cDNA coding for pea chloroplastic carbonicanhydrase. Plant Physiol 95: 264–268

Matsuda Y, Colman B (1995) Induction of CO2 and bicar-bonate transport in the green alga, Chlorella ellipsoidea: I.Time course of induction of the two systems. PlantPhysiol 108: 247–252

Matsuda Y, Hara T, Colman B (2001) Regulation of theinduction of bicarbonate uptake by dissolved CO2 in themarine diatom, Phaeodactylum tricornutum. Plant Cell En-viron 24: 611–620

Mitsuhashi S, Miyachi S (1996) Amino acid sequence ho-mology between N- and C-terminal halves of a carbonicanhydrase in Porphyridium purpureum, as deduced fromthe cloned cDNA. J Biol Chem 271: 28703–28709

Mitsuhashi S, Mizushima T, Yamashita E, Yamamoto M,Kumasaka T, Moriyama H, Ueki T, Miyachi S, Tsuki-hara T (2000) X-ray structure of �-carbonic anhydrasefrom the red alga, Porphyridium purpureum, reveals anovel catalytic site for CO2 hydration. J Biol Chem 275:5521–5526

Moroney JV, Bartlett SG, Samuelsson G (2001) Carbonicanhydrases in plants and algae. Plant Cell Environ 24:141–153




Moroney JV, Chen ZY (1998) The role of the chloroplast ininorganic carbon uptake by eukaryotic algae. Can J Bot76: 1025–1034

Moroney JV, Somanchi A (1999) How do algae concentrateCO2 to increase the efficiency of photosynthetic carbonfixation? Plant Physiol 119: 9–16

Nimer NA, Brownlee C, Merrett MJ (1999) Extracellularcarbonic anhydrase facilitates carbon dioxide availabilityfor photosynthesis in the marine dinoflagellate Prorocen-trum micans. Plant Physiol 120: 105–111

Nimer NA, Iglesias-Rodriguez MD, Merrett MJ (1997)Bicarbonate utilization by marine phytoplankton species.J Phycol 33: 625–631

Park YI, Karlsson J, Rojdestvenski I, Pronina N, KlimovV, Oquist G, Samuelsson G (1999) Role of a novelphotosystem II-associated carbonic anhydrase in photo-synthetic carbon assimilation in Chlamydomonas rein-hardtii. FEBS Let 444: 102–105

Price GD, Badger MR (1989) Expression of human car-bonic anhydrase in the cyanobacterium SynechococcusPCC7942 creates a high CO2-requiring phenotype. PlantPhysiol 91: 505–513

Price GD, Howitt SM, Harrison K, Badger MR (1993)Analysis of a genomic DNA region from the cyanobac-terium Synechococcus sp. strain PCC7942 involved in car-boxysome assembly and function. J Bacteriol 175:2871–2879

Raven JA (1997) CO2-concentrating mechanisms: a directrole for thylakoid lumen acidification? Plant Cell Envi-ron 20: 147–154

Rawat M, Moroney JV (1991) Partial characterization of anew isoenzyme of carbonic anhydrase isolated fromChlamydomonas reinhardtii. J Biol Chem 266: 9719–9723

Roberts SB, Lane TW, Morel FMM (1997) Carbonic anhy-drase in the marine diatom Thalassiosira weissflogii (Bacil-lariophyceae). J Phycol 33: 845–850

Rotatore C, Colman B (1991) The acquisition and accumu-lation of inorganic carbon by the unicellular green algaChlorella ellipsoidea. Plant Cell Environ 14: 377–382

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular clon-ing: A Laboratory Manual, Ed 2. Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY

Satoh A, Iwasaki T, Odani S, Shiraiwa Y (1998) Purifica-tion, characterization and cDNA cloning of soluble car-

bonic anhydrase from Chlorella sorokiniana grown underordinary air. Planta 206: 657–665

Smith KS, Ferry JG (1999) A plant-type (�-class) carbonicanhydrase in the thermophilic methanoarchaeon Meth-anobacterium thermoautotrophicum. J Bacteriol 181:6247–6253

Smith KS, Ferry JG (2000) Prokaryotic carbonic anhy-drases. FEMS Microbiol Rev 24: 335–366

Smith KS, Jakubzick C, Whittam TS, Ferry JG (1999)Carbonic anhydrase is an ancient enzyme widespread inprokaryotes. Proc Natl Acad Sci USA 96: 15184–15189

Strop P, Smith KS, Iverson TM, Ferry JG, Rees DC (2001)Crystal structure of the “cab” type beta class carbonicanhydrase from the archaeon Methanobacterium thermo-autotrophicum. J Biol Chem 276: 10299–10305

Sultemeyer D, Schmidt C, Fock HP (1993) Carbonic anhy-drases in higher plants and aquatic microorganisms.Physiol Plant 88: 179–190

Tachiki A, Fukuzawa H, Miyachi S (1992) Characteriza-tion of carbonic anhydrase isozyme CA2, which is theCAH2 gene product in Chlamydomonas reinhardtii. BiosciBiotechnol Biochem 56: 794–798

Tashian RE (1989) The carbonic anhydrases: widening per-spectives on their evolution, expression and function.Bioessays 10: 186–192

Tortell PD, Reinfelder JR, Morel FMM (1997) Active up-take of bicarbonate by diatoms. Nature 390: 243–234

Van K, Spalding MH (1999) Periplasmic carbonic anhy-drase structural gene (Cah1) mutant in Chlamydomonasreihardtii. Plant Physiol 120: 757–764

von Heijne G (1983) Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133: 17–21

Wilbur KM, Anderson NG (1948) Electrometric and col-orimetric determination of carbonic anhydrase. J BiolChem 176: 147–154

Williams TG, Colman B (1993) Identification of distinctinternal and external isozymes of carbonic anhydrase inChlorella saccharophila. Plant Physiol 103: 943–948

Yang SY, Tsuzuki M, Miyachi S (1985) Carbonic anhy-drase of Chlamydomonas: purification and studies on itsinduction using antiserum against Chlamydomonas car-bonic anhydrase. Plant Cell Physiol 26: 25–34

Satoh et al.



physiological and molecular biological characterization of ... · physiological and molecular...

Documents