From the Structural Biology Center, Korea Institute of Science and
Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea, the
§ Department of Biotechnology, Korea University, and the
Department of Chemistry and E. O. Lawrence Berkeley
National Laboratory, University of California,
Berkeley, California 94720
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ABSTRACT |
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Aquaspillium arcticum is a
psychrophilic bacterium that was isolated from arctic sediment and
grows optimally at 4 °C. We have cloned, purified, and characterized
malate dehydrogenase from A. arcticum (Aa MDH). We also
have determined the crystal structures of apo-Aa MDH, Aa MDH·NADH
binary complex, and Aa MDH·NAD·oxaloacetate ternary complex at
1.9-, 2.1-, and 2.5-Å resolutions, respectively. The Aa MDH sequence
is most closely related to the sequence of a thermophilic MDH from
Thermus flavus (Tf MDH), showing 61% sequence identity and
over 90% sequence similarity. Stability studies show that Aa MDH has a
half-life of 10 min at 55 °C, whereas Tf MDH is fully active at
90 °C for 1 h. Aa MDH shows 2-3-fold higher catalytic
efficiency compared with a mesophilic or a thermophilic MDH at the
temperature range 4-10 °C. Structural comparison of Aa MDH and Tf
MDH suggests that the increased relative flexibility of active site
residues, favorable surface charge distribution for substrate and
cofactor, and the reduced intersubunit ion pair interactions may be the
major factors for the efficient catalytic activity of Aa MDH at low temperatures.
Psychrophiles grow at low temperatures, where most of other
organisms cannot grow. In order to survive such extreme environments (less than 4 °C), enzymes from psychrophiles must catalyze
efficiently at low temperatures (1-6). While good progress is being
made to elucidate the adaptation mechanism of enzymes from some
extremophiles including hyperthermophile, the molecular basis of cold
adaptation of psychrophilic enzymes is relatively poorly understood
(7-9). However, psychrophilic enzymes have generated considerable
interest, since they can be used to improve the efficiency of
industrial processes and for environmental applications (1, 10).
Also, comparison of the structures of psychrophilic enzymes with
mesophilic, thermophilic, and hyperthermophilic counterparts may
add new insights into the understanding of catalytic mechanism and
analysis of thermostability factors.
As a first step to understand the structural basis of cold adaptation
of psychrophilic enzymes, we have carried out biochemical and
structural studies of malate dehydrogenase from Aquaspillium arcticum, a psychrophilic bacterium that was isolated from Arctic sediments and grows optimally at 4 °C (11).
MDH1 is a homodimeric enzyme
that catalyzes the reversible oxidation of malate to oxaloacetate in
the presence of NAD in the citric acid cycle and thus plays a major
role in central metabolism (12). Therefore, a certain amount of MDH is
always expected to be present in most living organisms. Several MDHs
from different sources have been extensively studied in genetic and
biochemical aspects; sequences of a large number of malate
dehydrogenases from organisms representing Archea, Bacteria, and
Eukarya have been reported, and many of their gene products have been
characterized. Furthermore, crystal structures of MDHs from the
thermophile Thermus flavus (13), the mesophile
Escherichia coli (14, 15), porcine heart mytochondria (16),
and cytoplasm (17) have been determined.
Recently, a few psychrophilic enzymes including subtilisin from
Bacillus TA41 (3), amylase from Alteromonas
haloplanctis A23 (4), citrate synthase from DS2-3R (5), and
alcohol dehydrogenase from Moraxella sp. TAE123 (6) have
been isolated, and their characteristics have been compared with
mesophilic or thermophilic counterparts. The commonly observed
biochemical features of these cold active enzymes are (i) their
increased catalytic efficiencies at low temperatures and (ii)
significantly increased thermolability compared with mesophilic or
thermophilic counterparts. However, it is difficult to understand the
properties of cold active enzymes at the molecular level from the
studies described above, since most of these studies were carried out
in the absence of structures of psychrophilic enzymes.
In the present study, we have cloned a MDH gene from A. arcticum and purified and characterized its product. We also
report here the high resolution crystal structures of apo-Aa MDH, Aa MDH·NADH binary complex, and Aa MDH·NAD·oxaloacetate
ternary complex (Table I) and describe our analysis of how
psychrophilic MDH may catalyze efficiently at low temperatures.
Purification--
Four liters of A. arcticum cells,
obtained from DSM, were grown in tryptic soy broth (Difco) at 4 °C
for 3-4 days. Cells were harvested and resuspended in 100 mM of Tris-Cl, pH 8.2, buffer containing 0.5 mM
phenylmethylsulfonyl chloride. The cells were lysed in a French press,
and the insoluble debris was removed by centrifugation. The supernatant
was loaded to a Q-Sepharose column equilibrated with 50 mM
Tris-HCl (pH 8.2). Fractions containing MDH were eluted between 0.4 and
0.5 M NaCl. These fractions were pooled, concentrated using
ultrafiltration, and applied to a Blue Sepharose CL-6B column
preequilibrated with same buffer. Aa MDH was eluted with 2 mM NADH. The protein was further purified by a size
exclusion column (Superdex 75). SDS-polyacrylamide gel electrophoresis
analysis of the final preparation showed a single band of protein
with 95% homogeneity.
Cloning--
Trypsin digestion of a purified Aa MDH results in
two fragments with approximate molecular mass of 27 and 9 kDa.
N-terminal sequencing of intact Aa MDH and a fragment corresponding to
27 kDa provides the sequences AKTPMRVAVTGAAGQLXYSLL and
XDLXIXXQIFTVQGXAXDAVA, respectively. Two degenerate oligomers were derived from these sequences, and polymerase chain reaction was performed using these primers and genomic DNA from A. arcticum as a template. The
polymerase chain reaction resulted in a major single product with the
expected size and several nonspecific products.
The genomic DNA was digested by several restriction enzymes, blotted
onto a Hybond-N+ membrane, and analyzed by Southern hybridization using
the polymerase chain reaction-amplified product as a probe. A 2.4-kb
HindIII fragment that hybridized to the polymerase chain reaction product was isolated, ligated with pBluescript KS(+), and
transformed into E. coli DH5 Kinetics and Thermostability Measurements--
Kinetic
parameters were determined for Aa MDH, Tf MDH (Sigma), and MDH from
E. coli (Ec MDH; Sigma). Reaction mixtures containing 100 mM Tris-HCl (pH 7.5), 500 µM oxaloacetate,
and enzyme were incubated at 4, 10, and 37 °C. The NADH (5-200
µM final concentration) were added to the mixture, and
the amount of NAD produced was measured at various temperatures and
times. Km and kcat values
were determined by Lineweaver-Burk plots using the ENZFITTER (18)
data analysis program.
Thermostability was measured for Aa MDH, Ec MDH, and Tf MDH in buffer
containing 100 mM Tris-HCl, 500 µM
oxaloacetate, 100 µM NADH, pH 7.5. Each enzyme was
incubated at 55 °C for various times and then cooled on ice. The
residual enzyme activity was measured with 500 µM
oxaloacetate and 100 µM NADH at 37 °C using the
standard protocol described above.
Crystallization--
The enzyme was dialyzed against 50 mM Tris-HCl, pH 8.2, 100 mM NaCl. Equal volumes
of protein (10 mg/ml) and reservoir solution (100 mM
Tris-HCl, 400 mM sodium acetate, 35% polyethylene glycol 4000, pH 8.0) were mixed and equilibrated with 1 ml of reservoir solution at 18 °C using the hanging drop vapor diffusion method. Crystals with a size of 0.2 × 0.2 × 0.3 mm appeared within
4-10 days. NADH binding was carried out by transferring the crystal to
a reservoir solution containing 100 mM NADH for 2 days. To make the ternary complex, the crystal was initially soaked in reservoir
solution containing 100 mM NAD for 2 days, and then oxaloacetate was added to a final concentration of 200 mM
and incubated at 18 °C for 36 h. x-ray diffraction data were
measured to 1.9 Å for the apo-Aa MDH, 2.1 Å for the binary complex,
and 2.5 Å for the ternary complex. All data were measured on a
Mar30 image plate system mounted on a Rigaku RU-200 rotating anode
x-ray generator. Data for the apo-Aa MDH and binary complex were
measured at 18 °C, whereas the data for the ternary complex were
collected at Refinement and Model Building--
The structure of the
apoenzyme was solved by the molecular replacement method as implemented
in X-PLOR (20) using a search model built from Tf MDH, where side
chains of nonidentical residues were replaced by alanine or glycine and
the portions with inserted or deleted regions were removed. Rotation
and translation functions of the method gave a unanimous solution; the
highest peak from the translation search in the resolution range of
15-4 Å was 3 times higher than the mean value. The initial R-factor
was 48.15%. One round of rigid body refinement dropped the R-factor to
29.7% in the resolution range of 10-4 Å. With a correctly positioned model, |Fo| Cloning and Sequence Comparison
Aa MDH is composed of 329 amino acids, and its sequence is most
similar to those of Tf MDH (61% identity and over 90% similarity) among all available MDH sequences (Fig.
1a). Composition analysis shows that Aa MDH has slightly lower Arg/(Arg + Lys) content (43%) than Tf MDH (51%). While Glu (13 versus 24%) and Arg (13 versus 19%) contents in Tf MDH are significantly higher
than those in Aa MDH, other charged residue contents are similar in two
enzymes. The numbers of glycine (28 versus 27 for Aa MDH
versus Tf MDH) and proline (16 versus 16)
residues are very similar in the two proteins. While numbers of
hydrophobic residues with the aromatic ring are equal in both MDH, some
differences are observed in isoleucine (23 versus 17),
valine (22 versus 27), and leucine (27 versus 30).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
. Sequencing of this plasmid confirmed the presence of a complete Aa MDH gene.
190 °C using a flash frozen crystal. Determination of
unit cell parameters and integration of reflections was performed using the program DENZO (19) from the HKL package. Data were scaled and
merged with SCALEPACK (19).
|Fc| and
2|Fo|
|Fc| maps were
calculated, and manual adjustment of the model was performed using the
program CHAIN (21). The model was further refined using X-PLOR with
Engh and Huber stereochemical parameters (22). Refinement statistics
are presented in Table I. Because the
first two N-terminal residues are not clearly defined, they are not included in the final model. The final models contain 327 residues and
220 water molecules for apo-Aa MDH; 327 residues, 127 water molecules,
and 1 NADH molecule for the binary complex; and 327 residues, 62 water
molecules, 1 NAD molecule, and 1 oxaloacetate molecule for the ternary
complex.
Diffraction data and refinement statistics
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
a, sequence alignment and position of
secondary structure elements in Aa MDH and Tf MDH. The single letter
code for amino acid residues is used. Every 10th residue is marked with
a filled circle. Residues interacting with
NAD/NADH and oxaloacetate are marked with asterisks.
b, ribbon diagram of the overall
structure of an Aa MDH subunit. Secondary structure labelings are
consistent with those in a. The N-terminal domain is in
violet, the C-terminal domain is in blue, NAD is
in green, and oxaloacetate is in yellow.
c, a stereodiagram of the quaternary structure of
Aa MDH. Each subunit is colored in red and
blue, NAD is in green, and oxaloacetate is in
yellow.
Kinetic and Stability Properties of Aa MDH
The kinetic constants were determined for MDHs from three species, a psychrophile, a mesophile, and a thermophile at three different temperatures, and are summarized in Table II. The kcat/Km of Aa MDH was approximately 2-3-fold higher than that of Ec MDH at 4-10 °C. The activities of Tf MDH at 4-10 °C were too low, and we could not measure the kinetic constant of Tf MDH at these temperatures within our limit. The kcat/Km values of Aa MDH and Ec MDH are almost equal at 37 °C.
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For stability studies, each enzyme was preheated at various temperatures for a given time, and the activities were measured. The half-time of inactivation (t1/2) of each MDH is shown in Table II. The activity of Aa MDH decreased to 50% at 55 °C for 10 min. The half-time of inactivation (t1/2) of Ec MDH was 20 min at 55 °C, showing a 2-fold increase compared with Aa MDH. Tf MDH retains full activity at 55 °C for more than 2 h. It has been reported that Tf MDH is fully active after heating at 90 °C for 1 h (13). Circular dichroism measurement also shows a large difference of Tm values between Aa MDH (58 °C) and Tf MDH (>95 °C).
X-ray Structure
Overall Structure--
We will focus on a structural comparison
between psychrophilic Aa MDH and thermophilic Tf MDH throughout this
study, since these two enzymes show remarkable sequence similarity
while having notably different biochemical properties. Each monomer of
homodimeric Aa MDH folds into two domains with different functions
(Fig. 1, b and c). The nucleotide binding domain
is formed from a twisted N-terminal six-stranded -sheet flanked by
-helices. The C-terminal catalytic domain consists of two
-sheets
and several helices. The active site is in a cleft between two domains.
The asymmetric unit contains an Aa MDH monomer, and the dimer has a
crystallographic 2-fold axis. The dimeric interface is formed from
helices
1,
2,
6, and
9-loop-
10 (Fig. 1c). As
expected from the high sequence similarity, the overall structure of Aa
MDH is very similar to that of Tf MDH, and it can be superimposed with
an r.m.s. deviation of 0.8 Å for 319 C-
atoms. The largest
differences are observed in the loop region between
10 and
10,
where the r.m.s. deviation value is 3.8 Å. Minor differences are
observed in the loop between
2 and
2, where two more residues are
inserted in Aa MDH. The Aa MDH and Ec MDH show more significant
deviations (r.m.s. deviation of 2.4 Å for 256 C-
atoms), reflecting
their low sequence identity of 27%.
NADH Binding Site--
The presence of NADH in the NADH-soaked
crystal was clearly identified from the simulated annealed omit map of
NADH complex crystal (Fig.
2a). Comparisons between
apo-Aa MDH and Aa MDH·NADH complex structures show an r.m.s.
deviation of 0.31 Å, suggesting that NADH did not induce any
noticeable conformational changes. The NADH is hydrogen-bonded to the
side chains of Glu43 (2.7, 2.9 Å), Gln115 (3.3 Å), Asn134 (3.5 Å), and His190 (3.0 Å) and
main chain atoms of Gly12 (3.2 Å), Gln16 (2.9 Å), Ile17 (2.9 Å), Val132 (3.0 Å), and
Asn134 (3.1 Å) (Fig. 2b). Most of the NADH
binding residues are highly conserved both in Aa MDH and Tf MDH (Fig.
1a). However, the side chain of Gln14 of Tf MDH
forms a hydrogen bond to the NO1 (3.3 Å) atom and AO-1 (3.1 Å) atom
of NADH, whereas the side chain of a corresponding residue,
Gln16 in Aa MDH points away from the NADH. These
differences may contribute to the increased Km of
NADH in Aa MDH compared with that in Tf MDH at 37 °C.
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It has been proposed that the surface loop formed by residues 90-100 plays an important role in the binding of NADH (13, 17). In lactate dehydrogenase, another NADH binding enzyme whose tertiary structure is similar to MDH, significant conformational changes in this loop occur upon binding of NADH (23, 24). While no direct evidence of such gross conformational changes within the surface loop has been observed in MDH, the compositions of the amino acids and the main chain atom positions in this region are very similar in both Aa MDH and Tf MDH.
Ternary Complex--
MDH catalyzes an ordered reaction, where NADH
binds first, followed by the dicarboxylic acid substrate (25). The
substrate binding site was identified by first soaking the crystal in
buffer containing NAD and later with oxaloacetate. The resulting omit map clearly reveals the presence of a NAD and a substrate (Fig. 3a). Although oxaloacetate
binds near NAD, no direct interactions were observed between the two
molecules. The C-4 atom of the nicotinamide moiety of NAD is 5.9 Å away from the C-2 atom of oxaloacetate, and a water molecule is present
between the two atoms (Fig. 3b). Important residues involved
in oxaloacetate binding include Arg165 (2.6 and 3.2 Å),
His190 (3.1 Å), and Ser241 (3.3 Å). The main
chain atoms of Arg229 (3.5 Å) and Gly230 (2.9 Å) also form hydrogen bonds to oxaloacetate (Fig. 3b). The carbonyl oxygen of Gly227 is 2.9 Å away from the O1A atom
of oxaloacetate. Thus, it is possible that these two atoms may form a
hydrogen bond if the O1A atom of oxaloacetate becomes protonated.
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In lactate dehydrogenase, the C-4 atom of the nicotinamide ring in NADH and the C-2 atom of the substrate analogue, oxamate, are located closer (3.7 Å), such that the proton can be directly transferred to the C-2 atom of lactate from NADH (23, 24). Structure comparison reveals that the binding of substrate to LDH induces a gross conformational change (up to 14 Å) in the surface loop around the NADH binding region, which is equivalent to residues 89-100 in Aa MDH. However, the binding of oxaloacetate to Aa MDH did not induce any notable conformational changes as judged by comparison of the ternary complex structure with apo- and binary Aa MDH enzyme structures (r.m.s. deviation of 0.33 and 0.34 Å, respectively). Therefore, it is possible that the large conformational change upon oxaloacetate binding may be necessary to bring the substrate closer to NAD, and this structural rearrangement is limited inside the crystal due to the crystal packing. Thus far, no ternary complex structures of MDH from other organisms, substrate, and coenzyme have been reported. However, the ternary complex of Ec MDH, NAD, and substrate analogue (citrate) has been determined at 1.9-Å resolution, and the distance between the C-4 atom of the nicotinamide ring in NAD and the C-3 atom of citrate was 4.9 Å (15).
Comparison of the oxaloacetate binding site of Aa MDH and an equivalent region of Tf MDH reveals that all of the residues interacting with oxaloacetate are conserved except for Gly227, which has been replaced by alanine in Tf MDH. Since the main chain of glycine has more conformational freedom than any other amino acid, substitution of Gly227 in Aa MDH from Ala227 may provide more local flexibility in Aa MDH, and this may partly contribute to the high catalytic efficiency of Aa MDH at low temperatures.
Flexibility--
It has been proposed that increased flexibility
is the most important factor for the catalytic efficiency of
psychrophilic enzymes at low temperatures (1, 2). The crystallographic thermal factors of the structures of Aa MDH and Tf MDH have been compared to analyze the flexibility of both enzymes. Aa MDH·NADH complex had an average B-factor of 15.10 Å2 for main chain
atoms, significantly lower than that of Tf MDH·NADH, 23.56 Å2. Also, average B-factors for main chain atoms of each
domain in Aa MDH (N-domain, 14.75 Å2; C-domain, 15.45 Å2) show similar differences compared with those in Tf MDH
(N-domain, 23.14 Å2; C-domain, 24.03 Å2).
However, differences in resolution, packing, solvent content, and
quality of data could contribute to B-factors. To correlate the local
flexibility of each MDH in an equivalent scale of thermal parameters,
we have divided the B-factors of all atoms in each enzyme by an average
B-factor of a whole molecule (termed relative B-factor) and compared
these values for the two proteins (Fig. 4). All main chain atoms
(Gly12, Gln16, Ile17,
Val132, Asn134; Gly227,
Arg229, Gly230) and most of the side chain
atoms (Glu43, Arg165, Ser241)
interacting with NADH and oxaloacetate in Aa MDH had up to
approximately 2-fold increased relative B-factors, reflecting their
increased relative flexibility (Fig. 4). We have also used two
different methods to compare the relative flexibility of the active
site regions in both MDHs. First, the B-factors for the atoms of active site residues were divided by the average B-factors of the rest of the
atoms in the whole molecule. This was to remove any bias resulting from
the active site residues in local flexibility calculation. Second, the
average B-factors for the whole molecule without the active site
residues were subtracted from the B-factors for each of the atoms in
the active site, and these values were divided by the S.D. values for
the average B-factors of the whole molecule without the active site
residues. In either case, the values that may represent the relative
local flexibility for the MDHs were higher in Aa MDH compared with Tf
MDH, suggesting that the active site in Aa MDH is relatively more
flexible (Fig. 4). The slightly increased flexibility in the regions of
NADH and oxaloacetate binding sites in Aa MDH may facilitate the
catalytic activity at low temperatures. Recent comparative studies of
citrate synthase from Antarctic bacterial strain DS2-3R and its
hyperthermophilic counterpart have shown that an overall average main
chain B-factor was much lower in psychrophilic citrate synthase
compared with that of hyperthermophilic enzyme (26). However, the small
domain in psychrophilic citrate synthase showed more flexibility
compared with the large domain, and this difference is more significant in the psychrophilic enzyme than that of its hyperthermophilic counterpart (26). Thus, the domain movement of citrate synthase for
enzymatic catalysis upon substrate binding at low temperature is more
favorable in psychrophilic citrate synthase. The hydrogen exchange
experiments also show evidence for an enhanced rigidity of thermophilic
proteins as compared with those from mesophilic proteins (27).
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Electrostatic Potential--
One of the most remarkable
differences between the structures of Aa MDH and Tf MDH is their charge
distributions in the accessible surface (28). As seen in Fig.
5, the surface around the NADH binding
region in Tf MDH is dominated by negative potentials, whereas that in
Aa MDH has significantly weaker negative potentials around the
corresponding region. The differences in the position of basic residues
of the surface loop comprising residues 91-100 (Arg91 and
Lys92 and Lys106 in Tf MDH/Arg93,
Arg95, and Lys110 in Aa MDH) results in the
different charge distributions within this loop (Fig. 5). The
oxaloacetate binding regions in each MDH also show some discrepancies
in charge distribution. The oxaloacetate binding region of Aa MDH has
more basic regions compared with Tf MDH, since Arg165,
His190, and Lys228 are present in Aa MDH,
whereas Arg161, His186, and Gln228
are in the equivalent region in Tf MDH.
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The increased positive potential at and around the oxaloacetate binding site and the significantly decreased negative surface potential at the NADH binding region in Aa MDH may facilitate the interaction of a negatively charged substrate toward the surface of the enzyme and may increase the catalytic efficiency at low temperature.
Thermolability-- It has been suggested that high flexibility of an enzyme is tightly correlated to the increased thermolability of the enzyme. Thus, we have analyzed the several factors that could contribute to enzyme stability. These include the number and location of proline/glycine residues and the number of inter- and intrasubunit ion pairs, hydrogen bonds, buried surface areas, and cavities (Table III).
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The numbers of glycine and proline residues are very similar in both MDHs, and the positions of these residues are highly conserved in Aa MDH and Tf MDH (Fig. 1a).
While the numbers of Asp, Lys, and His residues are similar, Tf MDH has
significantly more Glu and Arg than Aa MDH. Despite such differences,
Tf MDH has the same number of intrasubunit ion pairs compared with
those of Aa MDH (Table III). Also, an equal number of intrasubunit ion
pair networks are found in Aa MDH
(Arg24-Asp29-Lys33,
Glu319-Arg101-Glu320) and in Tf
MDH (Arg22-Glu27-Lys31,
Glu251-Arg156-Asp255). However,
Aa MDH has about half the number of intersubunit ion pairs as Tf MDH
(Fig. 6). In addition, only three
residues, A-Arg165, B-Asp62, and
A-Arg229 (A and B represent each subunit) form ion pair
networks, whereas five residue ion pair networks (B-Lys168,
A-Glu57, B-Arg229, A-Asp58, and
B-Arg161) are observed in Tf MDH.
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Ion pairs have emerged as a critical force in stabilizing hyperthermophilic enzymes (29-31). However, the relative importance of intra- or intersubunit ion pairs are still unclear. Also, the contribution of ion pairs to the stability of general proteins other than hyperthermophilic proteins requires further analyses. Nevertheless, the formation of an ion pair network is known to be important to the protein stability. The decreased number of intersubunit ion pairs and ion pair networks is probably one of the major forces in the thermolability of Aa MDH. Recent comparative studies of psychrophilic citrate synthase and its hyperthermophilic counterpart reveal that the psychrophilic enzyme has more intrasubunit and fewer intersubunit ion pairs, emphasizing the importance of intersubunit ion pairs and agreeing with our present analyses (26).
The numbers of hydrogen bonds in both MDHs show similar patterns of ion pairs; Aa MDH has more intrasubunit hydrogen bonds, whereas more intersubunit hydrogen bonds are found in Tf MDH .
The Aa MDH subunit has slightly lower accessible surface area compared with a subunit of Tf MDH (Table III). Aa MDH dimer has 116 Å of less buried surface area compared with that of Tf MDH. Considering that each square Å has about 25 cal/mol of energy gain, the decreased buried surface area of Aa MDH provides an ~2.9-kcal loss. The more significant difference is found in the nature of the buried surface area in each MDH. While the hydrophobic character is similar in both enzymes, the portion of charged residues in the buried surface area is remarkably smaller in Aa MDH (28%) compared with that of Tf MDH (37%). The increased charged character of the buried surface interface in Tf MDH results in the increased number of intersubunit ion pairs and charged polar group interactions. It has been proposed that the increased number of buried ion pairs in the protein interfaces could contribute to protein stability unlike those present in the core of a protein because of the differences in the surrounding environment and desolvation energy (32). Thus, slightly decreased buried surface area and a smaller portion of charged residues in dimeric interface may contribute to the increased thermolability of Aa MDH.
The number of cavities in both MDHs is the same, and the size of the cavities was very similar (33).
Conclusion
In this paper, we have reported the isolation and biochemical characterization of a MDH from the psychrophilic bacterium, A. arcticum, as well as three x-ray structures of Aa MDH. The psychrophilic MDH shows some interesting features: (i) it has 2-3-fold higher kcat/Km values compared with a mesophilic MDH at a temperature range of 4-10 °C; (ii) its primary structure is highly similar to that of a thermophilic MDH from Tf MDH; and (iii) Aa MDH shows significantly increased thermolability compared with Tf MDH.
Our comparative studies of MDHs from a psychrophile and a thermophile
point out three important factors for efficient catalysis by a
psychrophilic MDH at low temperatures: (i) an increased relative flexibility at and near the active site region of Aa MDH; (ii) more
positive potential in the surface around the oxaloacetate binding site
and decreased negative potential around the NADH binding site compared
with Tf MDH; and (iii) an increased thermolability of Aa MDH, which is
largely contributed by the significantly decreased intersubunit ion
pairs and buried surface area in the dimer.
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FOOTNOTES |
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* This work was supported by the MOST Biotech 2000 program, the Korea Institute of Science and Technology KIST 2000 program, and the U.S. Department of Energy.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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF109682.
The coordinates and structure factors (accession codes: 1b8p for apo-Aa MDH, 1b8v for Aa MDH·NADH, and 1b8u for Aa MDH·NAD·OAA) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ Supported by a KAST young scientist award in life science. To whom correspondence should be addressed. Tel.: 822-958-5937; Fax: 822-958-5939; E-mail: yunje{at}sbc4.kist.re.kr.
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ABBREVIATIONS |
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The abbreviations used are: r.m.s., root mean square; MDH, malate dehydrogenase; Aa MDH, A. arcticum malate dehydrogenase; Tf MDH, T. flavus malate dehydrogenase; Ec MDH, E. coli malate dehydrogenase.
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