Bageshwar, Umesh K.

Gokhman, Irena

Greenblatt, Harry M.

Lakshmanane, Prem

Savchenko, Umesh K.

Sussman, Joel L.

Zamir, Ada

University of Queensland eSpace

1084 Premkumar et al.  Halotolerant carbonic anhydrase Acta Cryst. (2003). D59, 1084±1086
crystallization papers
Acta Crystallographica Section D
Biological
Crystallography
ISSN 0907-4449
Identification, cDNA cloning, expression,
crystallization and preliminary X-ray analysis of an
exceptionally halotolerant carbonic anhydrase from
Dunaliella salina
Lakshmanane Premkumar,a,b
Harry M. Greenblatt,b Umesh K.
Bageshwar,a² Tatyana
Savchenko,a Irena Gokhman,a
Ada Zamira and Joel L.
Sussmanb*
aDepartment of Biological Chemistry,
Weizmann Institute of Science, Rehovot 76100,
Israel, and bDepartment of Structural Biology,
Weizmann Institute of Science, Rehovot 76100,
Israel
² Present address: Institute of Plant Genomics
and Biotechnology, Department of Horticulture,
Texas A&M University, College Station,
TX 77843, USA.
Correspondence e-mail:
joel.sussman@weizmann.ac.il
# 2003 International Union of Crystallography
Printed in Denmark ± all rights reserved
An extracellular -type carbonic anhydrase (dCAII) from the salt-
tolerant alga Dunaliella salina differs from its mesophilic counter-
parts in remaining active from zero to multimolar salt concentrations.
To gain insight into the outstanding salt tolerance of dCAII, the
enzyme was functionally overexpressed in Escherichia coli, puri®ed
by af®nity chromatography and crystallized by the hanging-drop
method. The crystals belonged to space group P21, with unit-cell
parameters a = 47.0, b = 119.9, c = 58.5 AÊ , = 94.2. Data from a single
crystal were collected to 2.4 AÊ resolution under cryogenic conditions
(120 K) using an R-AXIS IV++ detector mounted on a Rigaku
RU-H3R rotating-anode generator. The asymmetric unit contains
two molecules of the protein, which corresponds to VM =
2.65 AÊ 3 Daÿ1 and a solvent content of 52.7%.
Received 5 February 2003
Accepted 26 March 2003
1. Introduction
Proteins normally function within a narrow
range of relatively low salt concentrations. In
contrast, halophilic proteins derived from
extreme halophilic archaea usually require
multimolar salt concentrations for correct
folding and activity (Madern et al., 2000;
Mevarech et al., 2000).
Recently, members of another class of
proteins, designated as halotolerant, were
identi®ed in the extremely salt-tolerant
unicellular green alga Dunaliella salina
(Gokhman et al., 1999). D. salina cells grow in
salinities ranging from <0.1 M to near satura-
tion of NaCl, maintain a low intracellular ionic
concentration and achieve osmotic balance by
intracellular accumulation of glycerol (Avron,
1986). Hence, the intracellular proteins are not
challenged by salt, while the extracellular
proteins are expected to remain active under
widely differing salinities, extending over the
ranges suitable for mesophilic and halophilic
proteins.
The ®rst identi®ed protein ful®lling this
expectation was dCAI (originally named Dca),
a 60 kDa protein containing two linked repeats
of -type carbonic anhydrase sequences
(P54212). dCAI exhibits enzymatic activity
over the range from 0 to >2.0 M NaCl. In
composition, dCAI differed from other -type
carbonic anhydrases in containing an excess of
acidic over basic amino-acid residues (Fisher et
al., 1996).
Recently, we identi®ed in D. salina an
additional -type carbonic anhydrase: the
30 kDa non-duplicated dCAII. This enzyme is
57% identical to each N and C repeat of
dCAI and is likewise extracellularly oriented,
highly acidic and functionally salt tolerant
(Premkumar et al., 2003).
Towards the elucidation of the structural
elements underlying the unusual salt responses
of the D. salina carbonic anhydrases, we set out
to determine the three-dimensional structure
of dCAII. Here, we report the initial detection,
cDNA cloning, heterologous expression, puri-
®cation, functional characterization, crystal-
lization and preliminary X-ray analysis of
dCAII. Structural comparisons of dCAII with
numerous structures determined for meso-
philic carbonic anhydrases will help to eluci-
date the principles that underlie the high
degree of salt tolerance of the D. salina
enzyme.
2. Experimental and results
2.1. Detection and cDNA cloning of dCAII
Af®nity chromatography on a column
of agarose-coupled p-aminomethylbenzene
sulfonamide (Sigma) (Whitney, 1974) of
extracts of D. salina grown in 3.5 M NaCl
(Fisher et al., 1994; Sadka et al., 1989) yielded a
fraction eluted with 500 mM NaClO4 at pH 5.6
that predominantly contained the 60 kDa
dCAI (Bageshwar et al., unpublished results),
consistently accompanied by a minor 30 kDa
protein (dCAII). After its elution from an
SDS±PAGE gel, dCAII was subjected to
partial digestion with V8 protease and the
N-terminal sequence of several peptides was
determined as described in Fisher et al. (1996).
A sequence of 48 amino-acid residues,
compiled from two overlapping peptides,
showed homology to -type carbonic anhy-
drases and served to design forward and
Acta Cryst. (2003). D59, 1084±1086 Premkumar et al.  Halotolerant carbonic anhydrase 1085
crystallization papers
reverse degenerate primers. These were
used in RT-PCR (reverse transcriptase
polymerase chain reaction) together with
total RNA isolated from cells grown in
3.5 M NaCl as a template to amplify a 131 bp
cDNA sequence. 30 and 50 RACE (rapid
ampli®cation of cDNA sequence ends)
procedures were subsequently employed to
obtain the 1350 bp full-length cDNA that
encoded a protein of 317 amino-acid resi-
dues (dCAII).
Sequence alignments (Fig. 1) veri®ed the
identity of dCAII as an -type carbonic
anhydrase preceded by a leader peptide of
41 amino acids, of which 27 showed identity
to the N-terminal leader peptide of dCAI
(Fisher et al., 1996). Based on this alignment,
Glu41 in dCAII was assigned as the
N-terminal residue of the mature protein
(Fig. 1).
2.2. Plasmid construction, heterologous
expression and purification
The cDNA for mature dCAII (residues
41±317) was synthesized by PCR (30 cycles
at 368 K for 1 min, 327 K for 2 min and
345 K for 3 min) with the full-length cDNA
clone as template and the primers 50-GGA-
AGGATCCGAAGAACCCAACCCCAA-
TG-30 (forward) and 50-ATTGCTCGAG-
TTAATAGTTGTACTTGTACAC-30 (rev-
erse). The primers were designed to include
BamHI and XhoI restriction sites (in bold),
respectively. The PCR product was digested
with BamHI and XhoI and the 843 bp
product was inserted into the pET21a vector
(Novagen), pre-digested with BamHI and
XhoI, to create the expression vector
pdCAII. The insert was fused in-frame to
upstream vector sequences containing a
translation-initiation site followed by a
sequence encoding a T7 tag (see Novagen
catalogue for sequences). The recombinant
plasmid was transformed into the E. coli
Figure 1
The complete amino-acid sequence of dCAII aligned with N and C repeats of dCAI (P54212) and hCAI (human carbonic anhydrase isozyme I; P00915). The arrow shows the
N-terminus of mature dCAI after cleavage of the leader peptide (Fisher et al., 1996) and homology-based designation of the N-terminus of mature dCAII. The circled plus
signs label conserved His residues coordinating with Zn at the active site, ®lled circles label conserved Cys residues participating in disul®de-bond formation in extracellular
carbonic anhydrases and the empty circle labels the Cys75 residue in N-Dca that is probably involved in disul®de-bond formation with Cys262.
Figure 2
(a) Af®nity-puri®ed dCAII analyzed on SDS±PAGE (lane 2), size markers (lane 1). (b) Primitive monoclinic dCAI crystals grew in 2-propanol, PEG 4000 and trisodium
citrate pH 5.6 within 2±3 d and ranged from 0.1 to 0.8 mm in size. (c) Diffraction pattern extending beyond 2.4 AÊ of dCAII crystals. The rings from the centre correspond to
9.77, 4.88, 3.24 and 2.44 AÊ .
1086 Premkumar et al.  Halotolerant carbonic anhydrase Acta Cryst. (2003). D59, 1084±1086
crystallization papers
Origami B strain B(DE3)pLysS [(Fÿ ompT
hsdSB (rB
ÿmB
ÿ) lacY1 gor522::Tn10 (TcR)
trxB::kan (DE3) pLysS (CmR)]. Large-scale
expression of dCAII was carried out in a 12 l
culture grown in a fermentor, essentially as
described for dCAI (Premkumar et al.,
2003).
Af®nity puri®cation of recombinant
dCAII was carried out essentially as
described for recombinant dCAI
(Premkumar et al., 2003). Speci®cally, 10 g of
pelleted E. coli cells enriched in dCAII
was suspended in 40 ml lysis buffer and
lysed by repeated freezing in liquid nitrogen
and thawing in an ice-water bath as
described in Premkumar et al. (2003). The
lysate was spun at 42 000 rev minÿ1 for 1 h
and the supernatant was mixed with a
10 ml packed volume of agarose-bound
p-aminomethylbenzene sulfonamide pre-
equilibrated in 25 mM Na2SO4 in 25 mM
Tris±SO4 pH 8.8; the suspension was shaken
for 4 h at 277 K and poured into a column.
The bulk of the non-speci®cally bound
proteins were removed by washing with
300 ml equilibration buffer followed by
300 ml 100 mM NaClO4, 50 mM Na2SO4,
25 mM Tris±SO4 pH 8.8 (cf. Whitney, 1974).
The enzyme was eluted with 200 mM
NaClO4, 100 mM sodium acetate pH 5.6 as
described in Premkumar et al. (2003).
Complete EDTA-free protease-inhibitor
cocktail (Roche Molecular Biochemicals;
one tablet per 50 ml buffer solution) was
added to all solutions used. The eluted
fraction was extensively dialyzed against
25 mM Tris±SO4 pH 8.8, 10 mM ZnSO4 and
concentrated to 12.5 mg mlÿ1 in an
Amicon Centriprep YM-30. All puri®cation
steps were carried out at 277 K. SDS±PAGE
analysis of the puri®ed protein is shown in
Fig. 2(a).
The recombinant dCAII was assayed for
CO2 hydration and esterase activities in
increasing salinities. The results indicated
that both activities were retained from zero
to multimolar concentrations of NaCl (data
not shown). Similar results had previously
been observed for dCAI (Fisher et al., 1996;
Bageshwar et al., unpublished results).
2.3. Crystallization
Crystallization was performed at 292 K
using the hanging-drop vapour-diffusion
method. The crystallization conditions were
determined by a sparse-matrix screen using
Crystal Screen and Crystal Screen II kits
(Hampton Research). For the initial screen,
drops were prepared by mixing 1 ml of
protein solution (12.5 mg mlÿ1 in 10 mM
ZnSO4, 25 mM Tris±SO4 pH 8.8) with 1 ml
precipitant solution. The reservoir precipi-
tant volume was 0.5 ml.
A precipitate formed in Screen 41
(Crystal Screen), containing 20% 2-pro-
panol, 20% PEG 4000, 0.1 M trisodium
citrate pH 5.6, was the starting point for
optimization trials. Dilution of the precipi-
tant or lowering the protein concentration
produced clear drops or precipitates, but no
crystals. However, a variation on this screen
produced many beautiful albeit small crys-
tals (<0.1 mm) after 2±3 d by using the same
precipitant concentration in the reservoir
but preparing the drop with 1 ml protein
solution together with 1 ml of precipitant
which had been diluted 1:6 with water. When
the total drop size was increased to 6 ml,
retaining a 1:1 ratio of protein to diluted
precipitant (1:6), fewer but signi®cantly
larger crystals (0.1±0.7 mm) varying in size
and shapes (chunky, diamond, plates, rod)
were seen after 2±3 d (Fig. 2b).
2.4. Data collection
A native data set from a single chunky
crystal (0.2 mm in each direction) was
collected at 120 K with an R-AXIS IV
detector on an RU-300 rotating-anode
generator operating at 50 kV and 90 mA.
Each frame was recorded with a 10 min
exposure and 0.5 oscillation around ’. The
crystals were very sensitive to drop de-
hydration and layering oil onto the drop
containing crystals before manipulation was
crucial. The crystals were transferred to oil
and placed directly into a nitrogen stream
for data collection. The crystal-to-detector
distance was set to 200 mm so that the spots
were well resolved. The data set was inte-
grated and scaled using DENZO and
SCALEPACK (Otwinowski & Minor,
1997).
The recombinant dCAII crystallized in
space group P21, with unit-cell parameters
a = 47.0, b = 119.9, c = 58.5 AÊ ,  = 94.2. A
complete native data set for dCAII was
collected to a resolution of 2.4 AÊ (Fig. 2c)
and the statistics of the data processing are
shown in Table 1. Assuming that two 30 kDa
molecules are present in the asymmetric unit
and given a space group of P21, the
Matthews coef®cient (VM) was calculated to
be 2.65 AÊ 3 Daÿ1, with an estimated solvent
content of 52.7%.
Human carbonic anhydrases for which
crystal structures have been determined
(Stams & Christianson, 2000) share consid-
erable sequence identity with dCAII (37%
with the human carbonic anhydrase I).
However, using the known coordinates for
molecular replacement or treating the
template as polyalanine did not enable
solution of the structure of dCAII. Hence,
we plan to use the anomalous scattering by
the intrinsic catalytic Zn atom in dCAII to
carry out a MAD experiment to determine
the crystal structure of the protein. The
structure will be used in comparisons with
mesophilic counterparts in order to discern
for the ®rst time the distinctive structural
features conferring salt tolerance on an
-type carbonic anhydrase.
This study was supported by the Minerva
Foundation (Germany), the Nikken-
Sohonsha Corporation, Hashima City, Japan
and Nature Beta Technologies, Eilat, Israel.
JLS is the Morton and Gladys Pickman
Professor of Structural Biology.
References
Avron, M. (1986). Trends Biochem. 11, 5±6.
Fisher, M., Gokhman, I., Pick, U. & Zamir, A.
(1996). J. Biol. Chem. 271, 17718±17723.
Fisher, M., Pick, U. & Zamir, A. (1994). Plant
Physiol. 106, 1359±1365.
Gokhman, I., Fisher, M., Pick, U. & Zamir, A.
(1999). Microbiology and Biogeochemistry of
Hypersaline Environments, edited by A. Oren,
pp. 203±213. Boca Raton, FL, USA: CRC Press.
Madern, D., Ebel, C. & Zaccai, G. (2000).
Extremophiles, 4, 91±98.
Mevarech, M., Frolow, F. & Gloss, L. M. (2000).
Biophys. Chem. 86, 155±164.
Otwinowski, Z. & Minor, W. (1997). Methods
Enzymol. 276, 307±326.
Premkumar, L., Bageshwar, U., Gokhman, I.,
Zamir, A. & Sussman, J. L. (2003). Protein.
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Sadka, A., Lers, A., Zamir, A. & Avron, M. (1989).
FEBS Lett. 244, 93±98.
Stams, T. & Christianson, D. W. (2000). EXS, pp.
159±174.
Whitney, P. (1974). Anal. Biochem. 57, 467±476.
Table 1
Diffraction data.
Values in parentheses are for the highest resolution shell.
Crystal system Monoclinic
Space group P21
Crystal-to-detector distance (mm) 200.2
Data sweep () 126
Unit-cell parameters (AÊ , ) a = 47.0, b = 119.9,
c = 58.5,  = 90,
 = 94.2,  = 90
Matthews coef®cient (AÊ 3 Daÿ1) 2.65
Unit-cell volume (AÊ 3) 328780
No. of molecules per unit cell 4
No. of molecules in the AU 2
Total No. of unique
re¯ections collected
24493
Overall redundancy 2.56
No. of observed re¯ections 165404
Wavelength used (AÊ ) 1.5418
Resolution range (AÊ ) 35.0±2.4 (2.49±2.4)
Completeness (%) 96.1 (85.8)
Rmerge 0.026 (0.036)
hI/(I)i 46.6
Re¯ections <3 in highest
resolution shell
2.8
Mosaicity () 0.47


Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina

Lakshmanane Premkumar

Harry M. Greenblatt

Umesh K. Bageshwar

Tatyana Savchenko

Irena Gokhman

Ada Zamir

Joel L. Sussman

Crossref

Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from
                    Dunaliella salina

http://espace.library.uq.edu.au/view/UQ:247266/UQ247266_OA.pdf

Identification, cDNA cloning, expression, crystallization and preliminary X-ray analysis of an exceptionally halotolerant carbonic anhydrase from Dunaliella salina

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