From the Department of Biological Science and
Technology, Faculty of Engineering, University of Tokushima, Tokushima
770-8506, Japan, the ¶ Institute for Health Sciences, Tokushima
Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan, the
** National Institute of Advanced Industrial Science and
Technology, Tsukuba, Ibaraki 305-8566, Japan, and the
Structural Biophysics Laboratory, RIKEN
Harima Institute, Hyogo 679-5148, Japan
Received for publication, December 6, 2002, and in revised form, January 14, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A gene encoding a
2-deoxy-D-ribose-5-phosphate aldolase (DERA)
homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix. The gene was overexpressed in Escherichia
coli, and the produced enzyme was purified and characterized. The
enzyme is an extremely thermostable DERA; its activity was not lost
after incubation at 100 °C for 10 min. The enzyme has a molecular
mass of ~93 kDa and consists of four subunits with an identical
molecular mass of 24 kDa. This is the first report of the
presence of tetrameric DERA. The three-dimensional structure of the
enzyme was determined by x-ray analysis. The subunit folds into an
Aldolases catalyze carbon-carbon bond formation and cleavage and
are attractive as synthetic catalysts due to their ability to produce
stereospecific carbohydrates (1). They are divided into two major
classes based on the mode of stabilization of reaction intermediates.
Class I and II aldolases employ a Schiff base mechanism (2, 3) and a
divalent metal ion for intermediate stabilization (4), respectively.
2-Deoxy-D-ribose-5-phosphate aldolase
(DERA1; EC 4.1.2.4) belongs
to the class I aldolases and catalyzes a reversible aldol reaction
between acetaldehyde and D-glyceraldehyde 3-phosphate to
generate 2-deoxy-D-ribose 5-phosphate. DERA is unique in
catalyzing the aldol reaction between two aldehydes as both the aldol
donor and acceptor components. Its broad substrate specificity is an
attractive characteristic for the production of a variety of
stereospecific materials (5). The enzyme has high potential utility as
an environmentally benign alternative to chiral transition metal
catalysis of the asymmetric aldol reaction (6). However, the practical
application of DERA is still not successful because of its low stability.
Since DERA was first described by Racker (7), who reported the presence
of DERA in mammalian tissue as well as in Escherichia coli
and Corynebacterium diphtheriae, a number of the enzymes from eucaryotes and bacteria have been studied and characterized (8,
9). Despite its wide distribution, the physiological role of the enzyme
still remains obscure: nothing is known to date about its role in
eucaryotes, whereas in bacterial cells, it has been proposed to
function in the catabolism of deoxyribonucleosides (10, 11). In
Bacillus cereus, the enzyme has been reported to play a key
role in the utilization of the pentose moiety of exogenous nucleosides
(12, 13). In Salmonella typhimurium and E. coli,
the gene encoding DERA belongs to the deo regulon that
contains four genes encoding enzymes involved in nucleoside catabolism
(11, 14). The presence of DERA has not been described so far in
Archaea, the third domain of life.
Recently, much attention has been paid to the isolation and
characterization of enzymes from hyperthermophilic Archaea because the
organisms have high potentiality as a new source of much more stable
enzymes than the counterparts of mesophiles. A gene (open reading frame
identification number APE2437) encoding a DERA homolog has been
identified in the aerobic hyperthermophilic Archaea A. pernix via genome sequencing (15). In preliminary studies, we performed the cloning and expression of APE2437 in E. coli.
However, no functional products could be obtained. We purified the
native enzyme from A. pernix; analyzed the N-terminal amino
acid sequence; and identified that TTG, which is present at 126 bp
upstream from the 5' terminus of the predicted open reading frame, is
the proper initial codon. In this study, we succeeded in the expression
of the gene and purification and characterization of A. pernix DERA. In addition, we determined the crystal structure of
the enzyme at 2.0-Å resolution and revealed that it has a unique
tetrameric structure that might contribute to the high stability. We
present here the first report of the characterization and crystal
structure of hyperthermophilic DERA. These are essential steps in the
effort to comprehend its function and stabilizing mechanisms and also to achieve the practical application of DERA.
Chemicals--
The pET-11a vector was obtained from Novagen
(Madison, WI). The E. coli strain
BL21-CodonPlusTM-RIL was purchased from Stratagene (La
Jolla, CA). 2-Deoxy-D-ribose 5-phosphate, triose-phosphate
isomerase, and glycerol-3-phosphate dehydrogenase were obtained from
Sigma (Osaka, Japan). Restriction enzymes were purchased from New
England Biolabs Inc. (Beverly, MA). KOD DNA polymerase was
obtained from Toyobo (Osaka). All other chemicals were
reagent-grade.
Organism and Growth Conditions--
The hyperthermophilic
Archaea A. pernix K1 (JCM 9820) was obtained from the
Japanese Collection of Microorganisms (Wako, Saitama, Japan). The
microorganism was cultured in the modified medium of Sako et
al. (16), consisting of natural seawater containing 5 g/liter
trypticase peptone, 3 g/liter yeast extract, and 0.76 g/liter
Na2S2O3 (pH 7.0 adjusted with 0.5 N NaOH). Cells were grown by shaking (100 rpm) in an air
bath rotary shaker at 90 °C in 700 ml of medium in a 2-liter flask.
After 24 h of cultivation, sterilized water (~100 ml) was
replenished to avoid evaporation of the medium, and cultivation was
continued for an additional 18 h. The cells were collected by
centrifugation (7000 × g, 15 min) and washed twice
with 3% NaCl solution. The washed cells were suspended in 10 mM Tris-HCl buffer (pH 8.0).
Purification of DERA from A. pernix K1--
Cells (~30 g (wet
weight) from a 7-liter culture) were disrupted by
ultrasonication. Cell debris was removed by centrifugation (15,000 × g, 20 min, 4 °C), and nucleic acids were removed by the addition of streptomycin sulfate to a final concentration of 1%,
followed by centrifugation (15,000 × g, 20 min,
4 °C). The resultant supernatant was used as the crude extract. DERA was partially purified from the crude extract. The entire operation was
done at room temperature (~25 °C). The crude extract (120 ml) was
placed on a DEAE-Toyopearl column (5 × 15 cm; TOSOH,
Tokyo, Japan) equilibrated with 10 mM Tris-HCl buffer (pH
8.0). After the column was washed with the same buffer, the enzyme was
eluted with a 1000-ml linear gradient of 0-0.5 M NaCl in
the same buffer. The active fractions were pooled, and the enzyme was
dialyzed against 10 mM Tris-HCl buffer (pH 8.0). Solid
(NH4)2SO4 was added to the enzyme
solution up to 20% saturation. The enzyme solution was loaded onto a
butyl-Toyopearl column (4 × 10 cm; TOSOH) that was previously
equilibrated with the buffer supplemented with 20%
(NH4)2SO4. After the column was
washed with the same buffer (~3 column bed volumes), the enzyme was
eluted with a 1000-ml linear gradient of 20 to 0%
(NH4)2SO4 in the same buffer. The active fractions were collected and dialyzed against 10 mM
Tris-HCl buffer (pH 8.0).
PAGE--
SDS-PAGE (12% acrylamide slab gel, 1 mm thick) was
performed following the procedure of Laemmli (17). The protein band was stained with Coomassie Brilliant Blue R-250.
N-terminal Amino Acid Sequencing--
Approximately 3 µg of
protein was subjected to SDS-PAGE, followed by electroblotting onto a
polyvinylidene difluoride membrane. The membrane was then stained with
Ponceau S and destained. A protein band was excised and subjected to
automated Edman degradation using a Shimadzu Model PPSQ-10 protein sequencer.
Overexpression and Purification of Recombinant Protein--
The
following set of oligonucleotide primers was used to amplify the DERA
gene fragment by PCR:
5'-TATATCATATGCCGTCGGCCAGGGATATAC-3', containing a unique
NdeI restriction site overlapping the 5'-initiation codon
(TTG has been changed to ATG), and
5'-TAATGGATCCTTAGACTAGGGATTTGAAGC-3', containing a unique
BamHI restriction site proximal to the 3'-end of the
termination codon. The chromosomal A. pernix DNA was
isolated as described (18) and used as the template. The amplified
0.7-kb fragment was digested with NdeI and BamHI
and ligated with the expression vector pET-11a linearized with
NdeI and BamHI to generate pEDERA. E. coli strain BL21-CodonPlusTM-RIL was
transformed with pEDERA. The transformants were cultivated at 37 °C
in 200 ml of medium containing 2.4 g of Tryptone, 4.8 g of
yeast extract, 1 ml of glycerol, 2.5 g of
K2HPO4, 0.76 g of
KH2PO4, and 10 mg of ampicillin until the
absorbance at 600 nm reached 0.6. Induction was carried out by the
addition of 0.1 mM
isopropyl- Determination of Enzyme Activity--
Assay for DERA activity
was conducted at 50 °C essentially as described by Wong et
al. (19). The activity was determined by measuring the oxidation
of NADH in a coupled assay with triose-phosphate isomerase and
glycerol-3-phosphate dehydrogenase. The assay mixture contained 100 mM imidazole HCl buffer (pH 6.5), 0.1 mM NADH,
0.2 mM 2-deoxy-D-ribose 5-phosphate, 11 units
of triose-phosphate isomerase (rabbit muscle), 4 units of
glycerol-3-phosphate dehydrogenase (rabbit muscle), and the enzyme
preparation. The absorbance of NADH was followed at 340 nm ( Molecular Mass Determination--
The molecular mass of the
purified enzyme was determined by analytical gel filtration on a
Superdex 200 column (2.6 × 62 cm; Amersham Biosciences)
pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.0)
containing 0.2 M NaCl. Gel filtration calibration kits (Amersham Biosciences) were used for molecular mass standards. The
subunit molecular mass of the purified enzyme was determined by
SDS-PAGE using eight marker proteins (New England Biolabs Inc.).
Stability, pH Optima, and Kinetic Parameters--
To determine
thermostability, the enzyme solutions (0.5 mg/ml) in 10 mM
Tris-HCl buffer (pH 8.0) were incubated at different temperatures, and
the residual activity was determined by the standard assay method. To
determine pH stability, the enzyme (0.5 mg/ml) in buffers of various pH
values was incubated at 50 °C for 60 min, and the remaining
activities were then assayed. The buffers (500 mM) used
were acetate buffer, imidazole HCl buffer, Tris-HCl buffer,
glycine/NaOH buffer, and KCl/NaOH buffer at pH ranges of 3.5-6.0,
6.0-7.5, 7.5-8.5, 8.5-11.0, and 11.0-12.0, respectively. The
effects of organic solvents on enzyme stability were examined by
measuring the activity remaining after incubation with the reagents. An
aliquot of the incubation mixture was withdrawn, and the remaining
activity of the enzyme was assayed. The water-miscible organic solvents
used were methanol, ethanol, Me2SO, and
N,N-dimethylformamide. The optimal pH
of the enzyme was determined by running the standard assay at 50 °C
using citrate buffer (0.1 M), imidazole HCl buffer (0.1 M), Tris-HCl buffer (0.1 M), and glycine/NaOH
buffer (0.1 M) at pH ranges of 5.0-6.5, 6.0-7.5,
7.5-8.5, and 8.5-10.0, respectively. The Michaelis constants
were determined from Lineweaver-Burk plots (21) of data obtained
from the initial rate of 2-deoxy-D-ribose 5-phosphate
cleavage at 50 °C.
Crystallization and Data Collection--
Crystals were obtained
using the hanging-drop vapor diffusion method, in which 2 µl of 20 mg/ml protein solution was mixed with an equal volume of mother liquor,
consisting of 0.7-1.2 M NaH2PO4,
250 mM K2HPO4, and 100 mM Na2HPO4/citrate buffer (pH 4.2).
Crystals were grown at 20 °C for 3 days. The crystal belongs to the
orthorhombic space group P21212 with
the following unit cell parameters: a = 75.3, b = 83.2, and c = 87.2 Å. Heavy atom derivatives were prepared by soaking the crystals in a reservoir solution containing 1 mM thimerosal (10 h) or 1 mM K2Pt(SCN)6 (3 h). Data were
collected using an ADSC Quantum4R CCD detector system (Area Detector
Systems) on the BL-6A beamline at the Photon Factory in Tsukuba, Japan
(see Table I). All diffraction measurements were carried out on
crystals cryoprotected with ethylene glycol and cooled to 100 K in a
stream of nitrogen gas. The native and derivative data were processed
and integrated by DPS/mosflm (22) and scaled by scala (23).
Phasing and Refinement--
Native, mercury, and platinum data
sets (2.0-Å resolution) were used for phase calculation (Table
I) by the MIRAS (multiple isomorphous replacement with an
anomalous scattering) method using SOLVE (24).
The MIRAS map at 2.0 Å was subjected to maximum-likelihood density
modification, followed by autotracing using RESOLVE (25). An initial
model was built using Xtalview (26). The model was adjusted in Xtalview
using both |Fo| Identification of the Gene Encoding DERA--
The complete
sequence of the genome of A. pernix has been reported by
Kawarabayasi et al. (15). APE2437 (582 bp, positions 1,543,210-1,544,351 on the entire genome) has been annotated as the
gene encoding a putative DERA (a protein of 193 amino acids with a
molecular mass of 19,942 Da). The estimated molecular mass is obviously
smaller than that of the E. coli DERA (28 kDa). We performed
the cloning and expression of APE2437 in E. coli. However, no functional products could be obtained (data not shown). The activity
of DERA in the cell extract of A. pernix was observed to be
~0.0026 units/mg. The enzyme was purified 46-fold, and the specific
activity was 0.12 units/mg. The final preparation still contained
several contaminating proteins on the basis of SDS-PAGE analysis. The
N-terminal amino acid sequence of the protein that appeared as a major
band was analyzed, and the sequence of the protein with a molecular
mass of ~24 kDa was determined to be PSARDILQQG. On the basis of the
sequence, the upstream region of the 5' terminus of APE2437 was
analyzed. As a result, TTG, which was present at 126 bp upstream from
the 5' terminus of the predicted open reading frame, was identified to
be the proper initial codon for the DERA gene (Fig.
2). Kawarabayasi et al. (15)
assigned GTG as the start codon of this gene because sense codons
starting with ATG or GTG were used as the criteria for assignment of
the potential coding region in the genomic sequence. However, the
N-terminal analysis of the native enzyme shows that the sense codon of
the enzyme gene starts with a minor TTG codon, which is sometimes used
for the start codon in cyanobacteria (30). The newly identified gene
(708 bp) was estimated to code a protein of 235 amino acids with a
molecular mass of 24,529 Da. The
predicted amino acid sequence showed 28% identity to that reported for
E. coli DERA (Fig. 3) (9).
Expression of the Gene and Purification of the Recombinant
Enzyme--
E. coli strain BL21-CodonPlusTM-RIL
transformed with the expression vector pEDERA exhibited high activity
for DERA, which was not lost upon incubation at 100 °C for 10 min.
The enzyme was purified to homogeneity by heat treatment and two column
chromatographies from the extract of E. coli cells. An
efficient purification of the enzyme was achieved; ~180 mg of the
purified enzyme was obtained from 1 liter of E. coli
culture. The specific activity of the purified enzyme was estimated to
be 4.5 units/mg at 50 °C for the 2-deoxy-D-ribose
5-phosphate cleavage reaction.
Characteristics of A. pernix DERA--
The biochemical
characteristics of the purified enzyme were determined. SDS-PAGE of the
purified enzyme gave only one band; the subunit molecular mass was
determined to be ~24 kDa and was consistent with the molecular mass
calculated from the amino acid sequence (24,529 Da). The N-terminal
amino acid sequence was determined to be PSARDILQQGLD, which is
identical to that determined with the enzyme purified from A. pernix cells. This suggests that the first methionine is processed
in E. coli cells as well as in A. pernix cells.
The native molecular mass of the enzyme determined by gel filtration is
~93 kDa (Fig. 4). This indicates that
the enzyme consists of four subunits with identical molecular mass. E. coli DERA has a dimer structure (Protein Data Bank code
1JCL) composed of two identical subunits, which is most common for
DERA. The A. pernix enzyme is the first example of
tetrameric DERA.
The optimal pH for the 2-deoxy-D-ribose 5-phosphate
cleavage reaction was ~6.5. Typical Michaelis-Menten kinetics were
observed for the reaction at 50 °C. The apparent
Km value for 2-deoxy-D-ribose
5-phosphate was calculated to be 0.057 mM. The enzyme
retained full activity upon heating at 100 °C for 10 min and at
80 °C for 60 min (Fig. 5, A
and B). The thermostability of DERA has so far been reported
only for the E. coli enzyme. The enzyme is rapidly
inactivated above 70 °C (7). Thus, the A. pernix
enzyme is probably the most thermostable DERA among the enzymes from
other organisms described to date. The stability of the enzyme at
various pH values is shown in Fig. 5C. The enzyme was
extremely stable over a wide pH range; upon heating at 50 °C for 60 min, the enzyme did not lose activity at pH 4.5-11.0. The enzyme was
also highly resistant to organic solvents such as ethanol, methanol,
N,N-dimethylformamide, and
Me2SO at 50 °C. Loss of activity was not observed in the
presence of these reagents even at a concentration as high as 40%
(Fig. 5D). These results suggest that A. pernix
DERA might be preferred as a synthetic catalyst in practical
application.
Architecture of the Subunit--
The structure of A. pernix DERA was determined by the MIRAS method and refined at
2.0-Å resolution to an R-factor
(Rfree) of 22.4% (26.3%). The subunit
comprises 234 residues. The asymmetric unit consists of two homologous
subunits. The present model contains the complete ordered residues
2-235 of each subunit and 199 water molecules. The subunit folds into
an ( Monomeric Structural Comparison of A. pernix and E. coli
DERAs--
The structure of E. coli DERA was recently
reported (33). The structure of A. pernix DERA was compared
with that of E. coli DERA. The main chain coordinate of the
monomer of the A. pernix enzyme is quite similar to that of
the E. coli enzyme (root mean square deviation of 1.11 Å for the C- Oligomeric Structural Comparison of A. pernix and E. coli
DERAs--
The quaternary structure differs between the tetrameric
A. pernix and dimeric E. coli enzymes. The
functional tetramer (subunits A, B, C, and D) of the A. pernix enzyme is a ringed doughnut-like shape with approximate
dimensions of 82 × 70 × 50 Å (the dimensions of the hole
are 37 × 22 Å) (Figs.
8A and 9A). The A-B
and A-D association represents two distinct subunit-subunit interfaces. The two subunits of the A. pernix DERA asymmetric unit (A-B)
create an arrangement totally distinct from that observed for the
functional dimer of E. coli DERA (Fig. 8C).
The A-B interface is formed by the antiparallel alignment of the
Structural Features for Hyperthermostability--
Recent
structural studies on hyperthermophilic proteins reveal an increase in
the number of ion pairs and the formation of ion pair networks compared
with mesophilic counterparts (34-37). 168 and 177 ion pairs were
identified in the intrasubunits of A. pernix and
E. coli DERAs, respectively, using a cutoff distance between
oppositely charged residues of 3.0 Å. This indicates that there is no
significant difference in the number of total ion pairs and ion pair
networks in the intrasubunits between the two enzymes. In
general, the decrease in the solvent-accessible surface area and the
increase in the fraction of buried hydrophobic atoms have been
discussed as the stabilizing principles for thermostable protein (38).
As shown in Table II, the A. pernix DERA monomer (10,726 Å2) showed similar total
solvent-accessible surface area compared with the E. coli
enzyme monomer (10,751 Å2). No significant difference in
the number of hydrophobic residues (134 and 138 residues in A. pernix and E. coli DERAs, respectively) was observed
between the two enzymes. On the other hand, the area (4735 Å2) of the interface between subunits A, B, C, and D is
extremely larger than that (576.5 Å2) of the interface of
the E. coli enzyme dimer (Table II). The surface of the
inner section between A-D and B-C (A-B interface) and that between A-B
and D-C (A-D interface) are shown in Fig. 9 (B and C),
respectively. The green area (hydrophobic area) in the inner
section is remarkable compared with that in the outer section (Fig.
9D). This means that these tetrameric interactions are
mainly hydrophobic (green). The hydrophobic and ionic
residues involved in the A-B and A-D interactions are listed in
Table III. The hydrophobic interaction is especially striking
on the
A few enzymes from hyperthermophiles have been reported to assume a
higher oligomerization state than that of their mesophilic counterparts. These include Thermococcus kodakaraensis KOD1
ribulose-bisphosphate carboxylase/oxygenase (39) and
Thermotoga maritima dihydrofolate reductase (40) and
phosphoribosylanthranilate isomerase (41). The higher oligomerization
states of these proteins have been suggested to contribute to extreme
thermostability. However, the details of the relationship between the
oligomeric structure of these enzymes and thermostability are not still
clear. In this study, the structure of hyperthermophilic DERA was first
determined. The structure of aldolase from hyperthermophiles has not
been reported so far. This study suggests that the thermostability of
the aldolase can be enhanced by the formation of a unique quaternary structure unlike the mesophilic counterpart. In addition, thermostable DERA is expected to have high potentiality as a catalyst for the synthesis of some 2-deoxy-D-ribose 5-phosphate derivatives,
and the information on the three-dimensional structure in the active site may be useful for the development of this application.
/
-barrel. The asymmetric unit consists of two homologous
subunits, and a crystallographic 2-fold axis generates the functional
tetramer. The main chain coordinate of the monomer of the A. pernix enzyme is quite similar to that of the E. coli
enzyme. There was no significant difference in hydrophobic
interactions and the number of ion pairs between the monomeric
structures of the two enzymes. However, a significant difference in the
quaternary structure was observed. The area of the subunit-subunit
interface in the dimer of the A. pernix enzyme is much
larger compared with the E. coli enzyme. In addition, the
A. pernix enzyme is 10 amino acids longer than the E. coli enzyme in the N-terminal region and has an additional
N-terminal helix. The N-terminal helix produces a unique dimer-dimer
interface. This promotes the formation of a functional tetramer of the
A. pernix enzyme and strengthens the hydrophobic
intersubunit interactions. These structural features are considered to
be responsible for the extremely high stability of the A. pernix enzyme. This is the first description of the structure of
hyperthermophilic DERA and of aldolase from the Archaea domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside to the medium, and
cultivation was continued for 12 h at 18 °C. Cells (~50 g
(wet weight) from a 2-liter culture) were harvested by centrifugation,
suspended in 10 mM Tris-HCl buffer (pH 8.0), and disrupted
by ultrasonication. The crude extract was heated at 80 °C for 10 min, and the denatured protein was then removed by centrifugation
(15,000 × g, 20 min). Solid
(NH4)2SO4 was added to the enzyme
solution up to 20% saturation. The enzyme solution was loaded onto a
butyl-Toyopearl column and eluted using the same procedure as described
above. The active fractions were collected and dialyzed against 10 mM Tris-HCl buffer (pH 8.0), loaded onto a DEAE-Toyopearl
column, and eluted as described above. The active fractions were
pooled, dialyzed against 10 mM Tris-HCl buffer (pH 8.0),
and used as the purified enzyme preparation.
= 6.22 mM
1 cm
1). One enzyme unit
is defined as the amount catalyzing the cleavage of 1 µmol of
2-deoxy-D-ribose 5-phosphate/min. Protein was determined by
the method of Bradford (20) using the standard assay kit from Bio-Rad
with bovine serum albumin as the standard.
|Fc| and 2|Fo|
|Fc| maps.
Several cycles of rigid-body refinement, positional refinement, and
simulated annealing were performed at 2.0-Å resolution with CNS (27).
The final Rcryst and
Rfree were calculated to be 22.4 and 26.3%,
respectively (28). Fig. 1 shows the final
2|Fo|
|Fc| map. Model geometry was analyzed with PROCHECK (29), and 94.0% of the non-glycine residues were in the most favorable region of the Ramachandran plot and
6.0% in the additionally allowed region. The final coordinates have
been deposited in the Protein Data Bank (code 1N7K).
Statistics on data collection, phase determination, and refinement
=
=
= 90 °. Data were collected at the Photon
Factory on the BL-6A beamline using
= 1.00 Å.
Rsymn =
h
i|Ih,i
Ih
|/
h
iIh,i
Hg, 1 mM thimerosal (10 h), Pt, 1 mM
K2Pt(SCN)6 (3 h); FOM, figure of merit; r.m.s.d., root
mean square deviation.
View larger version (108K):
[in a new window]
Fig. 1.
The 2.0-Å resolution final
2|Fo| |Fc| map superimposed on the
refined 2.0-Å resolution coordinates of A. pernix DERA. The map was contoured at 1
.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 2.
Nucleotide sequence of the upstream region of
the 5' terminus of APE2437 and the deduced amino acid sequence.
The underlined sequence was determined from the N-terminal
region of DERA purified from A. pernix. The arrow
shows the predicted N-terminal amino acid sequence based on the
genome analysis.
View larger version (44K):
[in a new window]
Fig. 3.
Structure-based amino acid sequence alignment
of A. pernix and E. coli DERAs.
Sequences were aligned using ClustalX (42). -Helices
(
1-
10; green) and
-sheets
(
1-
8; yellow) are shown.
Asterisks represent conserved residues in the enzymes. The
residues involved in 2-deoxy-D-ribose 5-phosphate binding
in E. coli DERA are boxed.
View larger version (11K):
[in a new window]
Fig. 4.
Molecular mass of A. pernix
DERA. The molecular mass of the purified enzyme was
determined by analytical gel filtration on Superdex 200 as described
under "Experimental Procedures."
View larger version (29K):
[in a new window]
Fig. 5.
Effects of temperature, pH, and organic
solvents on A. pernix DERA stability.
A, after treatment at various temperatures for 10 min, the remaining activity was assayed at 50 °C. B, the
enzyme was incubated at 80 °C, and the activity of the sample was
assayed at 50 °C at appropriate intervals. C, the enzyme
in buffer of various pH values was incubated at 50 °C for 60 min,
and the remaining activity was then assayed at 50 °C. D,
the enzyme was incubated with various concentrations of water-miscible
organic solvents at 50 °C for 30 min. After incubation, the activity
of the aliquot was assayed at 50 °C. The organic solvents
used were ethanol ( ), methanol (
),
N,N-dimethylformamide (
), and
Me2SO (
).
/
)8-barrel carrying two additional helical
segments. The secondary structure was mapped onto the amino acid
sequence (Fig. 3), and the three-dimensional arrangement is shown in
Fig. 6. The elements of secondary
structure that create the barrel are
1-
3-
2-
4-
3-
5-
4-
6-
5-
7-
6-
8-
7-
9-
8-
10. The
2-helix caps the N-terminal section of the barrel
and is closely associated with the
10-helix, the
C-terminal helix of the barrel. The
1-helix forms an
"arm" protruding away from the barrel (Fig. 6), which
provides an important component for subunit-subunit interactions.
View larger version (56K):
[in a new window]
Fig. 6.
Crystal structure of A. pernix
DERA. The rainbow drawing shows the N terminus in
blue and the C terminus in red. The -helices
and
-sheets are numbered from the N terminus. The figure was
prepared using MOLSCRIPT (43) and Raster 3D (44).
atoms of 153 residues) (Fig.
7A). A noteworthy difference
between the two enzyme monomers is that the
1 arm is not
present in the E. coli enzyme. The structure of E. coli DERA has been determined in complex with
2-deoxy-D-ribose 5-phosphate (33). We predicted the
substrate-binding site by the superposition of A. pernix
DERA and the E. coli enzyme (Fig. 7B). Most of
the residues involved in substrate binding in E. coli DERA
(Asp16, Thr18, Asp102,
Lys137, Lys167, Thr170,
Gly171, Lys201, Gly204,
Gly205, Gly236, Ser238, and
Ser239) are conserved in the A. pernix enzyme,
except Cys47, Lys172, Val206, and
Arg234 are replaced with Val, Val, Ile, and Ile,
respectively (residue numbers of E. coli DERA are given). In
A. pernix DERA, Lys167 appears to play a
critical role in substrate binding because the same residue in the
E. coli enzyme forms a Schiff base with the substrate
(33).
View larger version (55K):
[in a new window]
Fig. 7.
A, stereographic drawing of the A. pernix DERA monomer structure (green) superimposed on
the E. coli DERA monomer structure (yellow). The
2-deoxy-D-ribose 5-phosphate molecules are represented by a
ball-and-stick model (red). The figure was prepared using
MOLSCRIPT (43) and Raster 3D (44). B, stereographic close-up
of the active site of A. pernix DERA. The
2-deoxy-D-ribose 5-phosphate of E. coli DERA was
built in for a better understanding (red). Highly conserved
residues in A. pernix and E. coli DERAs are shown
in green and are labeled. The figure was prepared using
Rasmol (32).
5-helices together with interactions with
residues in the N-terminal region of the
4-helix (Fig.
8B). The A-D interface is formed by the antiparallel
alignment of the
1-helices together with interactions
with the
9- and
10-helices (Fig.
8B). Neither the A-B nor A-D form is similar to the E. coli dimer. A. pernix DERA is 10 amino acids longer
than the E. coli enzyme in the N-terminal region, which
results in the presence of an additional N-terminal
1-helix (Fig. 3). This N-terminal helix might be
essential for the formation of the tetramer of A. pernix
DERA.
View larger version (38K):
[in a new window]
Fig. 8.
A, the functional A. pernix
DERA tetramer, with subunits A, B, C, and D shown in different colors.
B, the A-B and A-D interfaces. The 4- and
5-helices in the A-B interface and the
1-,
9-, and
10-helices in
the A-D interface, which are important for oligomerization, are
labeled. C, an arrangement of the two subunits of the
A. pernix DERA asymmetric unit (A-B; green) and
that observed for the functional dimer of E. coli DERA
(yellow). The A subunit of A. pernix DERA is
superimposed on the one subunit of the E. coli DERA
dimer. The figure was prepared using MOLSCRIPT (43) and Raster 3D
(44).
1-,
9-, and
10-helices in the A-D interface,
and these hydrophobic residues are Pro2 (
1),
Ile7 (
1), Leu8
(
1), Ile206 (
9),
Leu210 (
9), Leu234
(
10), and Val235 (
10). These
results suggest that the increase in intersubunit hydrophobic
interactions as a result of the formation of a tetramer plays important
roles in the extremely high stability of the A. pernix
enzyme. However, further study is required to prove this hypothesis.
Comparison of solvent-accessible surface areas
View larger version (48K):
[in a new window]
Fig. 9.
A, the solvent-accessible surface of the
functional A. pernix DERA tetramer, with subunits A, B, C,
and D labeled. The active sites are indicated by arrows.
B, the surface of the inner section between A-D and B-C (A-B
interface). C, the solvent-accessible surface of the inner
section between A-B and D-C (A-D interface). D,
the solvent-accessible surface of the outer section of the A-B dimer.
In B-D, the hydrophobic, acidic, and basic areas are shown
in green, red, and blue, respectively.
The figure was prepared using GRASP (31).
Hydrophobic and ionic residues involved in the interactions between the
A and B subunits and between the A and D subunits
![]() |
ACKNOWLEDGEMENTS |
---|
Data collection was performed at the Photon Factory. We thank Drs. M. Suzuki, N. Igarashi, and N. Sakabe (Photon Factory) for assistance with data collection.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the "National Project on Protein Structural and Functional Analysis" promoted by the Ministry of Education, Science, Sports, Culture, and Technology of Japan. Data collection performed at the Photon Factory was supported by the Tsukuba Advanced Research Alliance.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 1N7K) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Both authors contributed equally to this work.
Guest researcher in the Tsukuba Advanced Research Alliance.
§§ To whom correspondence should be addressed. Tel.: 81-88-656-7518; Fax: 81-88-656-9071; E-mail: ohshima@bio.tokushima-u.ac.jp.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212449200
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: DERA, 2-deoxy-D-ribose-5- phosphate aldolase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wong, C. H., Halcomb, R. L., Ichikawa, Y., and Kajimoto, T. (1995) Angew. Chem. Int. Ed. Engl. 34, 412-432 |
2. | Gefflaut, T., Blonski, C., Perie, J., and Willson, M. (1995) Prog. Biophys. Mol. Biol. 63, 301-340[CrossRef][Medline] [Order article via Infotrieve] |
3. | Morse, D. E., Tsolas, O., and Lai, C. Y. (1972) in The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. 7 , pp. 213-253, Academic Press, Inc., New York |
4. | Morse, D. E., and Horecker, B. L. (1968) Adv. Enzymol. 31, 125-181[Medline] [Order article via Infotrieve] |
5. | Barbas, C. F., III, Wang, Y. F., and Wong, C. H. (1990) J. Am. Chem. Soc. 112, 2013-2014 |
6. | Machajewski, T. D., and Wong, C. H. (2000) Angew. Chem. Int. Ed. Engl. 39, 1352-1375[CrossRef][Medline] [Order article via Infotrieve] |
7. | Racker, E. (1951) J. Biol. Chem. 196, 347-365 |
8. | Feingold, D. S., and Hoffee, P. A. (1972) in The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. 7 , pp. 330-321, Academic Press, Inc., New York |
9. | Valentin-Hansen, P., Boetius, F., Hammer-Jespersen, K., and Svendsen, I. (1982) Eur. J. Biochem. 125, 561-566[Abstract] |
10. | Munch-Petersen, A. (1970) Eur. J. Biochem. 15, 191-202[Medline] [Order article via Infotrieve] |
11. | Blank, J., and Hoffee, P. A. (1972) Mol. Gen. Genet. 116, 291-298[Medline] [Order article via Infotrieve] |
12. | Tozzi, M. G., Sgarrella, F., Barsacchi, D., and Ipata, P. L. (1984) Biochem. Int. 9, 319-325[Medline] [Order article via Infotrieve] |
13. | Sgarrella, F., Del Corso, A., Tozzi, M. G., and Camici, M. (1992) Biochim. Biophys. Acta 1118, 130-133[Medline] [Order article via Infotrieve] |
14. | Valentin-Hansen, P., Hammer-Jespersen, K., and Buxton, R. S. (1979) J. Mol. Biol. 133, 1-17[Medline] [Order article via Infotrieve] |
15. | Kawarabayasi, Y., Hino, Y., Horikawa, H., Yamazaki, S., Haikawa, Y., Jin-no, K., Takahashi, M., Sekine, M., Baba, S., Ankai, A., Kosugi, H., Hosoyama, A., Fukui, S., Nagai, Y., Nishijima, K., Nakazawa, H., Takamiya, M., Masuda, S., Funahashi, T., Tanaka, T., Kudoh, Y., Yamazaki, J., Kushida, N., Oguchi, A., Aoki, K., Kubota, K., Nakamura, Y., Nomura, N., Sako, Y., and Kikuchi, H. (1999) DNA Res. 6, 83-101[Medline] [Order article via Infotrieve], 145-152 |
16. | Sako, Y., Nomura, N., Uchida, A., Ishida, Y., Morii, H., Koga, Y., Hoaki, T., and Maruyama, T. (1996) Int. J. Syst. Bacteriol. 46, 1070-1077[Abstract] |
17. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
18. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , pp. 9.14-9.23, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
19. | Wong, C. H., Garcia-Junceda, E., Chen, L., Blanco, O., Gijsen, H. J. M., and Steensma, D. H. (1995) J. Am. Chem. Soc. 117, 3333-3339 |
20. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
21. | Cleland, W. W. (1971) in The Enzymes (Boyer, P. D., ed), 3rd Ed., Vol. 2 , pp. 1-65, Academic Press, Inc., New York |
22. | Rossmann, M. G., and van Beek, C. G. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 1631-1640[CrossRef][Medline] [Order article via Infotrieve] |
23. | Collaborative Computational Project Number 4. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve] |
24. | Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve] |
25. | Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 1863-1871[CrossRef] |
26. | McRee, D. E. (1992) J. Mol. Graphics 10, 44-46[CrossRef] |
27. | Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D. Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
28. | Brunger, A. T. (1992) Nature 335, 472-474[CrossRef] |
29. | Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef] |
30. | Sazuka, T., and Ohara, O. (1996) DNA Res. 3, 225-232[Medline] [Order article via Infotrieve] |
31. | Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[Medline] [Order article via Infotrieve] |
32. | Bernstein, H. J. (2000) Trends Biochem. Sci. 25, 453-455[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Heine, A.,
DeSantis, G.,
Luz, J. G.,
Mitchell, M.,
Wong, C. H.,
and Wilson, I. A.
(2001)
Science
294,
369-374 |
34. | Rice, D. W., Yip, K. S., Stillman, T. J., Britton, K. L., Fuentes, A., Connerton, I., Pasquo, A., Scandura, R., and Engel, P. C. (1996) FEMS Microbiol. Rev. 18, 105-117[CrossRef][Medline] [Order article via Infotrieve] |
35. | Knapp, S., de Vos, W. M., Rice, D., and Ladenstein, R. (1997) J. Mol. Biol. 267, 916-932[CrossRef][Medline] [Order article via Infotrieve] |
36. | Russell, R. J., Ferguson, J. M., Hough, D. W., Danson, M. J., and Taylor, G. L. (1997) Biochemistry 36, 9983-9994[CrossRef][Medline] [Order article via Infotrieve] |
37. | Hashimoto, H., Inoue, T., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., and Kai, Y. (1999) J. Mol. Biol. 292, 707-716[CrossRef][Medline] [Order article via Infotrieve] |
38. | Chan, M. K., Mukund, S., Kletzin, A., Adams, M. W., and Rees, D. C. (1995) Science 267, 1463-1469[Medline] [Order article via Infotrieve] |
39. |
Maeda, N.,
Kanai, T.,
Atomi, H.,
and Imanaka, T.
(2002)
J. Biol. Chem.
277,
31656-31662 |
40. | Dams, T., Auerbach, G., Bader, G., Jacob, U., Ploom, T., Huber, R., and Jaenicke, R. (2000) J. Mol. Biol. 297, 659-672[CrossRef][Medline] [Order article via Infotrieve] |
41. | Hennig, M., Sterner, R., Kirschner, K., and Jansonius, J. N. (1997) Biochemistry 36, 6009-6016[CrossRef][Medline] [Order article via Infotrieve] |
42. | Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G., and Gibson, T. J. (1998) Trends Biochem. Sci. 23, 403-405[CrossRef][Medline] [Order article via Infotrieve] |
43. | Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
44. | Merritt, E. A., and Murphy, M. E. P. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 869-873[CrossRef][Medline] [Order article via Infotrieve] |