From Biochemistry Option, California Institute of
Technology, Pasadena, California 91125, the § Department of
Biochemistry and Molecular Biology, The Pennsylvania State University,
University Park, Pennsylvania 16802, and the ¶ Howard Hughes
Medical Institute and Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, California 91125, and the
Center for Microbial Structural Biology, Pennsylvania State
University, University Park, Pennsylvania 16802
Received for publication, October 9, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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The structure of the "cab"-type Carbonic anhydrases
(CAs)1 are
Zn2+-containing enzymes that catalyze the reversible
hydration of CO2 (1). With turnover numbers approaching
106 s The The class
carbonic anhydrase from the archaeon Methanobacterium
thermoautotrophicum (Cab) has been determined to 2.1-Å
resolution using the multiwavelength anomalous diffraction
phasing technique. Cab exists as a dimer with a subunit fold similar to
that observed in "plant"-type
class carbonic anhydrases. The
active site zinc is coordinated by protein ligands Cys32,
His87, and Cys90, with the tetrahedral
coordination completed by a water molecule. The major difference
between plant- and cab-type
class carbonic anhydrases is in the
organization of the hydrophobic pocket. The structure reveals a Hepes
buffer molecule bound 8 Å away from the active site zinc, which
suggests a possible proton transfer pathway from the active site to the solvent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1, CAs are among the fastest
known enzymes. CAs have been found in most types of organisms,
including mammals, plants, algae, bacteria, and archaea (2). Based on
the amino acid sequences, CAs can be assigned to one of three
independently evolved classes, designated
,
, and
(3).
class contains all mammalian CAs, as well as some CAs from
algae and bacteria (3, 4).
-CAs play important roles in respiration,
secretion of HCO
-CA have revealed a
monomer organized around a 10-stranded, predominantly antiparallel
-sheet (7-13). The catalytically active zinc is coordinated by
three histidines and one water molecule.
-CA has thus far been isolated and characterized only from the
methanoarchaeon Methanosarcina thermophila (14-16), where
it is proposed to facilitate the transport of
CH3COO
and to convert CO2 to
HCO
-CA exists as a
trimer, with the active site located at the interface between two
subunits. Each subunit is organized around a left-handed
-helix that
is completely distinct from the
-CA fold, although the active site
is also coordinated by three histidines, along with two water molecules
(17, 18).
class includes CAs from plants, algae, bacteria, and archaea
(2, 3). In higher plants,
-CAs play an important role in
photosynthesis, by concentrating CO2 in the proximity of
ribulose bisphosphate carboxylase/oxygenase for CO2
fixation (19). The purification and characterization of carbonic
anhydrase (Cab) from the thermophilic Methanobacterium
thermoautotrophicum extends this class into the archaea (20). Cab
is at the phylogenetic extreme of the
class carbonic anhydrases and
forms an exclusively prokaryotic clade consisting primarily of
sequences from Gram-positive bacteria (2). In the obligate
chemolithoautotroph M. thermoautotrophicum, Cab converts
CO2 to HCO
class carbonic anhydrases can be further divided into "plant"-
and "cab"-type, based on the active site sequence conservation (20,
23) (Fig. 1). Two crystal structures of plant-type
-CA were
recently reported from Porphyridium purpureum
(P. purpureum
-CA) (24) and Pisum
sativum (P. sativum
-CA) (23). The basic fold of
-CA consists of a four-stranded, parallel
-sheet core with
-helices forming right-handed cross-over connections (23, 24). The
oligomerization state of
-CA is variable, however, and P. purpureum
-CA and P. sativum
-CA exist as a dimer
and octamer, respectively, although the dimer of P. purpureum
-CA resembles a tetramer, where two
monomers are fused together. In contrast to the protein ligation by
three histidines observed in
- and
-CAs, the active site zinc in
-CAs is coordinated by two cysteines and one histidine, as
anticipated from extended X-ray absorption fine structure spectroscopy
studies (21, 25, 26). The fourth ligand is different in the two
-CAs
structures. In the P. sativum
-CA structure, an acetate
molecule is bound to the zinc, whereas in the P. purpureum
-CA structure, the side chain of aspartic acid (Asp151)
acts as the fourth ligand. In the P. sativum
-CA
structure, this conserved Asp interacts with a conserved Arg (Fig.
1).
View larger version (38K):
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Fig. 1.
Alignment of -CA
sequences. Cab, M. thermoautotrophicum;
PPnterm, P. purpureum N-terminal domain;
PPcterm, P. purpureum C-terminal domain;
P.S., P. sativum; ECcynT, E. coli. Zinc ligands are colored yellow, the
conserved Asp/Arg pair is red, and residues differentiating
cab-type and plant-type are blue.
Apart from the conserved zinc ligands and the Asp/Arg pair, the active
site of Cab differs significantly from the plant-type -CAs. Cab
active site residues His23, Met33,
Lys53, Ala58, and Val72 are
replaced in the plant-type
-CA by Gln, Ala, Phe, Val, and Tyr,
respectively. Residues that are equivalent to Cab residues Met33, Lys53, Ala58, and
Val72 make up the hydrophobic pocket in the P. sativum and P. purpureum
-CA structures.
Substitutions of the two aromatic side chains by Lys and Val in Cab
suggest a significant redesign of the hydrophobic pocket in cab-type
-carbonic anhydrase. Cab has a CO2 hydration activity
with a kcat of 1.7 × 104
s
1 and Km for
CO2 of 2.9 mM at pH 8.5 and 25 °C (20). Cab
is inhibited by iodide, nitrate, and azide; however, in contrast to
plant-type
-CAs, chloride and sulfate have no effect on Cab activity. These active site substitutions, together with the different effects of inhibitors, imply that there might be mechanistically relevant differences in the organization of the active sites between cab-type and plant-type enzymes. Here we present the first structure of
the cab-type
-carbonic anhydrase from thermophilic methanoarchaeon M. thermoautotrophicum, determined at 2.1-Å resolution.
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EXPERIMENTAL PROCEDURES |
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Crystallization-- Cab was overexpressed in Escherichia coli and purified by a heat denaturation step followed by ion exchange chromatography as previously described (20). Crystals were grown by the hanging drop method at 22 °C using a 5 mg/ml protein solution and a precipitant solution containing 100 mM Hepes, pH 7.5, 35% ethanol, 12% 2-methyl-2,4-pentanediol, and 50 mM calcium acetate. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 54.9 Å, b = 113.2 Å, c = 156.2 Å and three dimers per asymmetric unit. Crystals were transferred stepwise to mother liquor solution containing 30% 2-methyl-2,4-pentanediol as a cryoprotectant and flash frozen.
Data Collection and Processing--
A three-wavelength
multiwavelength anomalous dispersion data set was collected at
160 °C on beamline 9-2 of the Stanford Synchrotron Research
Laboratory with an ADSC charge-coupled device detector. The
fluorescence spectrum measured around the zinc edge of a single crystal
was used to select the inflection point (
= 1.2832 Å), the
absorption edge (
= 1.282 Å), and a high energy remote
wavelength (
= 1.033 Å) for optimization of the anomalous signal. All data were reduced using DENZO and scaled using
SCALEPACK (27) (Table I).
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Phase Determination-- The structure was determined by multiwavelength anomalous dispersion using the signal from only the intrinsic zinc atoms. The program SOLVE (28) was used to find the positions of the heavy atoms using the three wavelength multiwavelength anomalous dispersion data set using data from 20- to 2.4-Å resolution. Two zinc ions were identified and when used for phasing yielded a figure of merit of 0.44. Four additional zinc sites were located in an anomalous difference Fourier map, yielding a figure of merit of 0.54. Each of the six zinc sites corresponded to a Cab monomer, which are organized into three tight dimers in the asymmetric unit. The initial noncrystallographic symmetry (NCS) transformations were established by the relationships between the dimers, and the initial mask was calculated with the program NCSMSK (29). The program DM (30) was used for NCS averaging of the electron density maps, solvent flattening, and phase extension from 2.4- to 2.1-Å resolution. The resulting map was of good quality and allowed building of most of the protein.
Refinement-- Alternate cycles of manual model building using the program O (31) and positional and individual B-factor refinement with the program CNS (32) reduced the R and Rfree to 21.1 and 24.6% respectively, where Rfree is calculated for 5% (2875) of the reflections in the resolution range 18-2.1 Å. The model was initially refined with strict NCS restraints, which were released later in the refinement. The r.m.s. deviation of bond lengths and angles are 0.013 Å and 1.7° respectively, with 87.9% in the most allowed region and 11.1% in the additionally allowed region of the Ramachandran plot. The average B-factors are 28.6 Å2 (main chain), 32.4 Å2 (side chains), 18.6 Å2 (zinc atoms), and 36.3 Å2 (solvent). An average B-factor of 30.5 Å2 is calculated for all protein atoms. The final model contains 7543 protein atoms, 3 Hepes molecules, 6 zinc atoms, 6 calcium atoms, and 409 water molecules, for a total of 8015 atoms (Table II).
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RESULTS AND DISCUSSION |
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Structural Organization of Cab--
The overall fold of the Cab
monomer (Fig. 2A) consists of
a four-stranded parallel -sheet core with strand order 2-1-3-4. Monomers in each dimer are related by a 2-fold axis centered between strands
2. The overall dimensions of the dimer are ~40 × 45 × 50 Å (Fig. 2B). Three dimers, designated AB, CD,
and EF, are present in the asymmetric unit. In the structurally
conserved
-sheet region (residues 26-32, 52-57, 80-88, 149-157,
and 163-167), the r.m.s. deviations in C
positions between the
dimers average 0.24 Å. Using C
positions for residues
24-170, the corresponding r.m.s. deviations are 0.34 Å between dimers
AB and CD, 1.0 Å between dimers AB and EF, and 1.1 Å between dimers
CD and EF. The relatively large r.m.s. deviations for the latter two
pairs of dimers reflect the poor ordering for residues 91-126 in
monomer E. Excluding these residues, the r.m.s. deviations drop to
~0.28 Å. The r.m.s. deviation in the
-sheet region between the A
and B, C and D, and E and F subunits of the Cab are ~0.6 Å, while
the r.m.s. deviation for all C
between the subunits are ~0.8
Å.
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The regions with the largest conformational variation include the
N-terminal residues 1-23 (involved in crystal packing), residues
92-95, and residues 120-125. While the N-terminal residues 1-23 are
well defined in monomers A, C, and E, residues 13-23 are disordered in
monomers B, D, and F. Residues 92-95 and 120-125 form a hinge region
for helices 4 and
5 and are well ordered in monomers B and D,
slightly disordered in monomers A, C, and F, and disordered in monomer
E. The variability in region 90-125 between the six
crystallographically independent monomers suggests that these residues
are conformationally mobile. Overall, with the exception of the noted
regions, the three dimers in the asymmetric unit are very similar.
Unless stated otherwise, only dimer AB will be used in future discussions.
Six calcium ions were located on the surface of Cab and at the crystal packing interfaces between Cab dimers. Most likely, these calcium ions do not play any structural or catalytical role and are a result of the crystallization conditions that contained 50 mM calcium acetate.
Cab Oligomerization--
-CAs have been found in different
oligomeric states ranging from dimers (Oryza
sativa, P. purpureum) to tetramers (E. coli) to octamers (P. sativum) (20, 23, 24, 33).
Although analytical ultracentrifugation results suggest that Cab exists
as a homotetramer (21), Cab appears to form a dimer in the crystal.
There are numerous interactions stabilizing the dimer involving
residues in
-strand
2 and helices
2,
3,
4, and
5.
Hydrogen bonds between residues 56 and 57 from strand
2 in both
subunits result in a formation of a 10-stranded
sheet. Helices
4
and
5 extend out and make extensive contacts with the other monomer
in the dimer (Fig. 2B). The interface area between these two
subunits was found to be ~2110 Å2/subunit, which is
~21% of the total (~10,000 Å2) monomer accessible
surface area as calculated with GRASP (34). Residues A2-A23 pack
against a symmetry-related molecule burying 860 Å2,
resulting in the formation of a continuous ribbon through the crystal
(Fig. 3). Residues B2-B12 most likely
fold back and pack against the B monomer in the same way that A2-A12
packs against the symmetry-related molecule. Residues B13-B23 have no
visible electron density and are disordered. A similar type of crystal packing forming a continuous ribbon has been observed in the sterile
motif domain of the human EphB2 receptor and, together with other
evidence, was proposed to be functionally important (35). The crystal
packing interaction seen in Cab can also be described as a linear
(open-ended) domain swapped oligomer, where the swapped domain consists
of a 12-residue
-helix (36, 37). It is unclear whether residues
1-24 could facilitate the formation of a higher oligomerization state
under physiological conditions or whether residues A2-A23 also fold
back and pack against monomer A. Based on modeling considerations,
although the N-terminal region is highly flexible, formation of a Cab
tetramer similar to the one seen in the P. purpureum
structure is unlikely in the absence of conformational changes due to
steric clashes of helices
4 and
5. Other contacts between Cab
dimers in the crystal are relatively small (
540 Å2) and,
based on their complementarity and size (38, 39), are also unlikely to
support formation of stable tetramers.
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Comparison of Cab and P. sativum and P. purpureum
Structures--
The fold of Cab is similar to that of the P. sativum and P. purpureum -CAs (Fig.
4). While the
-sheet core is
conserved, significant secondary structure differences are evident,
mostly in the regions at the N terminus, at the C terminus, and in the region containing residues 90-125. The N termini of the P. sativum and P. purpureum
-CA structures extend out,
forming a long helix that packs against a second monomer, making
additional dimer interactions. In Cab, the N terminus is involved in
crystal packing and does not adopt the same conformation observed in
the P. sativum and P. purpureum
-CA
structures. Cab is one of the smallest
-CAs known and lacks an
extended C terminus. In the P. sativum
-CA structure, the
C terminus forms a long
-strand that mediates octamerization. In
Cab, residues 90-125 form two helices (
4,
5) that project out to
cover the second monomer (Fig. 2B) and fold back to start
helix
6. In both the P. sativum and P. purpureum
-CA structures, this segment is longer, forms three
helices instead of two, and folds back earlier to create two additional
turns of helix
6. In the central
-sheet region, the r.m.s.
deviations in C
positions between Cab and P. sativum and
P. purpureum structures are 0.62 and 0.56 Å, respectively.
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Active Site--
The active site cleft is located at the C
terminus of the parallel -sheet and is largely sequestered from
solvent. Each Cab subunit contains one zinc atom that resides at the
interface of the two monomers (Fig. 2B), although the
coordination residues (Cys32, Cys90, and
His87) originate from the same monomer. One water molecule
completes the tetrahedral coordination sphere of the zinc. Although the crystallization conditions contained 50 mM calcium acetate,
no acetate was found in the active site, unlike in the P. sativum
-CA structure (Fig.
5A). The average coordination
distances from the six active sites are as follows: Cys32
S
-Zn (2.42 ± 0.03 Å), His87 N
-Zn
(2.11 ± 0.04 Å), Cys90 S
-Zn (2.40 ± 0.04 Å), and H2O-Zn (2.15 ± 0.06 Å). The four zinc ligands form a number of hydrogen bonds with the surrounding residues. His87 hydrogen-bonds with the carbonyl oxygen of residue
Thr88; Cys32 S
hydrogen-bonds with the amide
nitrogens of residues Lys22, Asp34, and
Gly59; Cys90 hydrogen-bonds with the amide
nitrogen of residue Met92; and the coordinating water
molecule makes a hydrogen bond to Asp34. Arg36
forms the conserved Asp/Arg pair with Asp34 and also
interacts with Asp89 and a water molecule.
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Rather unexpectedly, the two active sites A and B in the Cab dimer
exhibit significant differences, which are reflected in the r.m.s.
deviations between monomers in a dimer (~0.65 Å) being consistently
larger than between the equivalent monomers in different dimers such as
A and C, or B and D (0.24 Å). The active sites in monomers A, C, and E
have a Hepes buffer molecule bound near the active site. The sulfate
group of the Hepes is located ~8 Å from the zinc atom, and the
sulfate oxygens hydrogen bond with Lys53 N,
Ser35 O
, and the amide nitrogen of
Ser35 (Fig. 5B). The equivalent to
Ser35 is present in both plant-type
-CAs and in Cab,
while Lys53 is unique to the cab-type
-CAs.
Asp34, which makes a hydrogen bond to the zinc-coordinating
water molecule, is also within hydrogen bonding distance (~3.0 Å) to
the Hepes sulfate group. Another difference between the two active
sites is the conformation of residues 13-24. In the active site of
subunit A, residues B13-B24 are disordered and have no visible
electron density. In the active site of subunit B, however, residues
A13-A24 are well defined and extend out to participate in crystal
packing. Superposition of residues in the active sites of subunits A
and B (Fig. 5B) indicates that the Hepes molecule would
sterically clash with residues Arg16 and Asp17
if residues 13-24 adopted the same conformation as in active site B.
Comparison of P. sativum -CA and Cab Active Sites--
The
zinc-coordinating residues (Cys32, His87, and
Cys90) of Cab and P. sativum
-CA superimpose
closely (Fig. 6A). The water
molecule serving as the fourth zinc ligand in Cab adopts a position
similar to the O1 of the acetate ligand in the P. sativum
-CA structure. The conserved residue Asp34 is held in
place by forming two hydrogen bonds to Arg36, as do the
corresponding residues Asp162 and Arg164 in the
P. sativum
-CA. The following five active site
substitutions distinguish the cab-type and plant-type
-carbonic
anhydrases: H23Q, M33A, K53F, A58V, and V72Y. The superposition of Cab
and P. sativum
-CA structures clearly shows the
distinctions in active site organization by these residues.
His23, found on a flexible loop, is ~25 Å away from the
catalytic zinc and probably is not a part of the active site. On the
other hand, the equivalent residue Gln151 in the P. sativum
-CA structure occupies a position similar to the Hepes
sulfate group that is ~8 Å away from the zinc atom and is possibly
involved in ligand binding (23). The hydrophobic pocket of Cab is quite
different from that of plant-type
-CA (Fig. 6A). In
P. sativum
-CA structure, the hydrophobic pocket is
formed by Phe179, Val184, and
Tyr205. The corresponding residues in Cab
(Lys53, Ala58, and Val72)
constitute a more open and less hydrophobic pocket.
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Comparison of P. purpureum -CA and Cab Active Sites--
Again,
the protein ligands to the zinc in the two structures superpose closely
(Fig. 6B). The fourth ligand is different, since the side
chain of Asp151 in the P. purpureum
-CA
structure coordinates the zinc instead of the water molecule seen in
Cab structure. Asp151 of P. purpureum
-CA is
equivalent to Asp34 of Cab. As a consequence of the zinc
coordination by Asp151 in the P. purpureum
-CA structure, this residue cannot pair with the conserved Arg, and
Arg153 has flipped away from the active site. The adjacent
Ser152 has moved by ~4 Å and adopts a different
conformation in the P. purpureum
-CA structure (Fig.
6B). The hydrophobic pocket arrangement of P. purpureum
-CA is very similar to that of P. sativum
-CA, and the differences between the cab-type and plant-type hydrophobic pocket have already been discussed.
Mechanism--
The catalytic mechanism of carbonic anhydrases has
been most extensively studied for the -CA class (5, 6, 40-42). The zinc hydroxide mechanism established for this class provides an appropriate framework for discussing the catalytic mechanism of Cab. In
the first part of the CO2 hydration reaction,
CO2 binds in the hydrophobic pocket and probably interacts
with the amide nitrogen of Thr199. This threonine is known
as the "gatekeeper," and the side chain plays an important role in
the
-CAs, together with Glu106, in orienting the
CO2 molecule for attack by the zinc-bound hydroxide. In the
P. sativum
-CA structure, Asp162,
Gly224, and Gln151 are thought to play the same
role in orienting CO2 for this attack (23). In Cab,
Asp34 and Gly91 are in the same orientation as
Asp162 and Gly224 in P. sativum
-CA structure and might also help to orient CO2. His23, the equivalent of P. sativum
-CA
Gln151, is, however, disordered in active sites A, C, and
E, and in active sites B, D, and F it is at the beginning of a segment
of residues that pack against the symmetry-related molecule and lies 25 Å away from the active site zinc.
The second, rate-limiting, step in the CO2 hydration
reaction involves the regeneration of a hydroxide ion from the
zinc-bound water molecule. In -CAII, the zinc ion is located in a
deep funnel and requires a proton shuttle to transfer the proton to the
bulk solvent. His64 of
-CAII adopts multiple
conformations, which facilitates accepting the proton from the
zinc-bound water molecule and delivering it to buffer in bulk solution
(43). In
-CA, Glu84 exhibits multiple conformations and
has been proposed to participate in a proton shuttle (18, 44). Residues
with multiple conformations have not been described in the active site
of any
-CA structure determined so far. Since the
-CA active site
is closer to the surface of the protein than the
-CA active site, a
protein-mediated proton shuttle might not be necessary. The
-CA
reaction rate depends on buffer concentration, implying that proton
transfer can be rate-limiting under certain conditions (21). In the Cab structure, a Hepes buffer molecule was found near the active sites A,
C, and E. The Hepes sulfate group is located ~8 Å away from the zinc
atom and lies within hydrogen bonding distance of Asp34,
which makes a hydrogen bond to the zinc bound water molecule. In the
P. purpureum
-CA structure, the equivalent of
Asp34 acts as the fourth zinc ligand, and in the proposed
mechanism it plays a role in the proton transfer (24). Therefore, the most plausible pathway for proton transfer in Cab is from the zinc-bound water molecule to Asp34 and then to the sulfate
group of the bound Hepes molecule or a solvent molecule. The
conformation of residues 1-25 in active sites B, D, and F is
incompatible with Hepes binding, and residues 13-25 must adopt
different conformations for Hepes to bind (Fig. 5B). The
mobility of residues 1-25 and 92-125 might allow buffer molecules to
diffuse into the active site and serve as the proton acceptor necessary
to regenerate the zinc-bound hydroxide.
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ACKNOWLEDGEMENTS |
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We thank Jessica Wuu and Radu Georgescu for help with crystallizations and Brian R. Crane and Alex M. Bilwes for help with data collection.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM44661 (to J. G. F) and GM45162 (to D. C. R.); a National Science Foundation predoctoral fellowship (to P. S.); and NASA-AMES Cooperative Agreement NCC2-1057 (to the Pennsylvania State University Astrobiology Research Center). This work is based upon research conducted at the Stanford Synchrotron Radiation Laboratory, which is funded by the Department of Energy (Office of Basic Energy Sciences and Office of Biological and Environmental Research) and the National Institutes of Health (National Center for Research Resources, NIGMS).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G5C) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** To whom correspondence should be addressed: Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Code 147-75CH, Pasadena, CA 91125. Tel.: 626-395-8393; Fax: 626-744-9524; E-mail: dcrees@caltech.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009182200
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ABBREVIATIONS |
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The abbreviations used are:
CA, carbonic
anhydrase;
Cab, M. thermoautotrophicum class carbonic
anhydrase;
-CA,
class carbonic anhydrase;
r.m.s., root mean
square;
NCS, noncrystallographic symmetry.
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