Báo cáo khoa học: Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex doc

Crystal structure of archaeal highly thermostable
L-aspartate dehydrogenase/NAD/citrate ternary complex
Kazunari Yoneda
1
, Haruhiko Sakuraba
2
, Hideaki Tsuge
3
, Nobuhiko Katunuma
3
and Toshihisa Ohshima
1
1 Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
2 Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan
3 Institute for Health Sciences, Tokushima Bunri University, Japan
In prokaryotes, de novo NAD biosynthesis generally
proceeds via a condensation reaction between l-aspar-
tate and dihydroxyacetone phosphate that is catalyzed
by two enzymes: l-aspartate oxidase (LAO; the nadB
gene product) and quinolinate synthase (QS; the nadA
gene product) [1]. LAO catalyzes the oxidation of
l-aspartate to iminoaspartate, after which QS catalyzes
the condensation of iminoaspartate with dihydroxyace-
tone phosphate to produce quinolinate. Quinolinate is
then converted to nicotinate mononucleotide by quino-
linate phosphoribosyltransferase (the nadC gene prod-
uct), which is followed by conversion to NAD via a
metabolic sequence involving two enzymes: nicotinate
mononucleotide adenylyltransferase and NAD syn-
thase [2].
We recently detected the presence of LAO in Pyro-

coccus horikoshii OT-3, an anaerobic hyperthermo-
philic archaeon that grows optimally at 98 °C [3]. In
addition to the oxidase reaction, this LAO also cata-
lyzed FAD-dependent l-aspartate dehydrogenation
using fumarate as an electron acceptor. This was the
first example of an LAO produced in either the archa-
eal domain or an obligate anaerobic organism. There-
after, we identified genes encoding homologs of four
other enzymes involved in de novo NAD biosynthesis
in the P. horikoshii genome and found that nadB forms
Keywords
aromatic pair interaction; hyperthermostable
L-aspartate dehydrogenase; ion-pair
interaction; NAD biosynthesis
Correspondence
T. Ohshima, Institute of Genetic Resources,
Faculty of Agriculture, Kyushu University,
Hakozaki, Fukuoka 812-8581, Japan
Fax: +81 92 642 3059
Tel: +81 92 642 3053
E-mail:
(Received 13 May 2007, revised 26 June
2007, accepted 28 June 2007)
doi:10.1111/j.1742-4658.2007.05961.x
The crystal structure of the highly thermostable l-aspartate dehydrogenase
(l-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglo-
bus fulgidus was determined in the presence of NAD and a substrate ana-
log, citrate. The dimeric structure of A. fulgidus l-aspDH was refined at
a resolution of 1.9 A
˚

with a crystallographic R-factor of 21.7% (R
free
¼
22.6%). The structure indicates that each subunit consists of two domains
separated by a deep cleft containing an active site. Structural comparison
of the A. fulgidus l-aspDH ⁄ NAD ⁄ citrate ternary complex and the Thermo-
toga maritima l-aspDH ⁄ NAD binary complex showed that A. fulgidus
l-aspDH assumes a closed conformation and that a large movement of the
two loops takes place during substrate binding. Like T. maritima l-aspDH,
the A. fulgidus enzyme is highly thermostable. But whereas a large number
of inter- and intrasubunit ion pairs are responsible for the stability of
A. fulgidus l-aspDH, a large number of inter- and intrasubunit aro-
matic pairs stabilize the T. maritima enzyme. Thus stabilization of these
two l-aspDHs appears to be achieved in different ways. This is the first
detailed description of substrate and coenzyme binding to l-aspDH and
of the molecular basis of the high thermostability of a hyperthermophilic
l-aspDH.
Abbreviations
LAO,
L-aspartate oxidase; L-aspDH, L-aspartate dehydrogenase; MIRAS, multiple isomorphous replacement with anomalous scattering;
QS, quinolinate synthase.
FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4315
an operon with nadA, nadC and three other unknown
genes. We therefore proposed that a de novo NAD bio-
synthetic pathway functions in P. horikoshii under
anaerobic conditions [3]. More recently, a previously
unknown amino acid dehydrogenase, l-aspartate dehy-
drogenase (l-aspDH; the TM1643 gene product),
was identified in a hyperthermophilic bacterium Ther-
motoga maritima by Yang et al. based on the 3D struc-

ture of the gene product [4]. This enzyme catalyzes
NAD(P)-dependent dehydrogenation of l-aspartate to
produce iminoaspartate. Because the gene encoding
l-aspDH also forms an operon with nadA and nadC
within the T. maritima genome, those authors sug-
gested that l-aspDH catalyzes the first step in the
de novo biosynthesis of NAD in this organism. Thus,
two different types of oxidoreductase may have
evolved to catalyze the first step of de novo NAD
biosynthesis in prokaryotes [4]. Bearing in mind
that hyperthermophilic archaea are phylogenetically
ancient, our interest in the phylogenetic relationship
between LAO and l-aspDH led us to screen for an
l-aspDH homolog in the genomes of hyperthermo-
philic archaea. Within the genomic sequence of an
anaerobic hyperthermophilic archaeon, Archaeoglobus
fulgidus, we found a gene (AF1838) whose predicted
amino acid sequence exhibits 38% identity with that of
T. maritima l-aspDH. In addition, we showed that the
gene product expressed in Escherichia coli acts as a
highly thermostable l-aspDH [5].
Yang et al. determined the structure of T. maritima
l-aspDH in the presence of NAD and analyzed the
structural features responsible for its dehydrogenase
activity [4]. T. maritima l-aspDH does not share struc-
tural similarity with the superfamilies that include the
l-leucine, l-valine, l-glutamate and l-phenylalanine
dehydrogenase, and l-alanine dehydrogenase, despite
all these enzymes catalyzing similar chemical reactions.
In addition, the structure of T. maritima l-aspDH has

only low similarity to aspartate semialdehyde dehydro-
genase, inositol 1-phosphate synthase and dihydrodipi-
colinate dehydrogenase. This enzyme thus appears to
represent a new class of amino acid dehydrogenase.
However, details about the molecular strategy underly-
ing its high thermal stability, as well as the manner in
which substrate and coenzyme are bound by the
enzyme, are still unclear. Therefore, our aim was to
determine the structure of the A. fulgidus l-aspDH in
complex with NAD and a substrate analog, citrate.
Factors that could stabilize the enzyme were then com-
pared with those in the T. maritima l-aspDH, and the
structural features that appear to be responsible for
the high thermostability of each enzyme are discussed.
Finally, we describe a substrate-induced conforma-
tional change in the ternary complex of A. fulgidus
l-aspDH.
Results and Discussion
Overall structure
The structure of A. fulgidus l-aspDH was determined
using multiple isomorphous replacement with anoma-
lous scattering (MIRAS) and was refined at a resolu-
tion of 1.9 A
˚
(Table 1). The asymmetric unit consisted
of one homodimer with a solvent content of 39.3%,
which corresponds to a Matthew’s coefficient [6] of
2.0 A
˚
3

ÆDa
)1
. The model contained 236 ordered amino
acid residues in each subunit and 136 water molecules.
The two almost identical (r.m.s.d. ¼ 0.42 A
˚
) subunits
had approximate dimensions of 35 · 53 · 44 A
˚
and
were related by a twofold noncrystallographic rotation
axis and associated closely through antiparallel b sheets
(b11 and b11¢) (Fig. 1A). Each monomer consisted of
two domains: an N-terminal coenzyme-binding domain
(a1–a5, a10, b1–b6, b12–b14), which formed a classical
Rossmann-fold motif, and a C-terminal domain ( a6–
a9, b7–b11), in which both the catalytic activity and
formation of the homodimer were mediated (Fig. 1B).
Overall domain organization was similar to that of
T. maritima l-aspDH, but with several critical differ-
ences in the structural details.
Structural comparison of A. fulgidus and
T. maritima
L-aspDHs
When we compared the structures of the A. fulgidus
l-aspDH ⁄ NAD ⁄ citrate ternary complex and the
T. maritima l-aspDH ⁄ NAD binary complex, we found
that the overall folds of A. fulgidus and T. maritima
l-aspDH were similar (Fig. 3A), which was expected,
given their relatively high sequence identity (38%)

(Fig. 2). The internal structure of the A. fulgidus
enzyme was basically the same as that of the T. mari-
tima enzyme (r.m.s.d. ¼ 1.9 A
˚
for the Ca atoms of 223
residues), although a marked difference was observed
in the position of three loops in the catalytic domain.
When the Rossmann-fold domain of the A. fulgidus
l-aspDH structure was superimposed on that of the
T. maritima l-aspDH structure, we observed a large
shift in the positions of two loops (loop 1: R133–
G143, loop 2: V180–I188 in the A. fulgidus l-aspDH)
toward the active site cavity (Fig. 3). The average
movements of loops 1 and 2 were estimated to be 3.2
and 3.6 A
˚
, and the largest movements of loops 1 and 2
were estimated to be 6.7 A
˚
(K142) and 5.8 A
˚
(E183), respectively. As described below, this large
Crystal structure of L-aspDH from A. fulgidus K. Yoneda et al.
4316 FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS
conformational change may be essential for substrate
binding. In addition, loop 3 (P208–S216), which is dis-
ordered in the T. maritima enzyme, was clearly obser-
vable in the structure of A. fulgidus enzyme and
formed a flap over the active site cavity (Fig. 3). It has
been proposed that the structure of T. maritima l-as-

pDH assumes an open configuration [4]. Our results
strongly suggest that loop 3 functions as the substrate-
binding site and the structure of A. fulgidus l-aspDH
assumes a closed configuration.
Substrate binding
In the electron-density map of A. fulgidus l-aspDH
obtained from our preliminary experimental data, we
noticed an extra density within the active site cavity
and found that a citrate molecule could be modeled
into that density after construction and refinement of
the peptide chain. The map clearly defined the precise
orientation of the citrate (Fig. 4A): the two oxygen
atoms in the C-1 carboxyl group of the citrate form
hydrogen bonds with the side chain of K134, a proton
at the main chain amide of L161, and a water mole-
cule (WAT33); two oxygen atoms in the C-5 carboxyl
group are located within hydrogen-bonding distance of
the side chain of N162 and backbone amide protons in
N162 and V163; oxygen atoms in the C-6 carboxyl
group form hydrogen and ionic bonds with the side
chains of N187, H189 and S216, and the main chain
amide proton in S216; and an oxygen atom in the C-3
hydroxyl group forms a hydrogen bond with the side
chain of K134. Together, these bonds tightly hold the
citrate near the nicotinamide ring of NAD. Based on
the structure of citrate, we modeled the l-aspartate
molecule into the active site of A. fulgidus l-aspDH
(Fig. 4B) and then minimized the energy of the com-
plex using insight ii (Biosym ⁄ MSI, San Diego, CA).
Within this structure, two oxygen atoms of the C a car-

boxylate are situated within hydrogen- and ionic-bond-
ing distance of the side chains of N187, H189 and
S216, and the main chain amide proton of S216. In
addition, the oxygen atoms of the Cb carboxyl group
Table 1. Statistics on data collection, phase determination and refinement. The crystal belongs to space group P2
1
2
1
2 with a ¼ 47.52 A
˚
,
b ¼ 89.58 A
˚
, c ¼ 100.49 A
˚
.
Native Hg1
a
Hg2
b
Hg3
c
Data collection
Maximum resolution (A
˚
) 1.9 2.0 2.0 1.79
Total reflections 237826 212964 202467 289592
Unique reflections 34 001 55997 54558 77004
Redundancy 7.0 3.8 3.7 3.8
Completeness

d
(%) 98.0 (87.6) 99.9 (99.1) 97.2 (85.6) 98.2 (86.7)
R
sym
d,e
(%) 5.5 (23.9) 6.4 (22.9) 5.1 (21.3) 5.4 (25.4)
<I⁄ r (I)>
d
15.7 (5.9) 13.0 (4.4) 13.7 (4.0) 15.7 (3.4)
MIRAS phasing
FOM 0.68 (32.6–2.0)
Refinement
Resolution range (A
˚
) 32.6–1.90
R
cryst
f
(%) 21.7
R
free
g
(%) 22.6
No. of protein atoms 3686
No. of water molecules 136
No. of NAD 2
No. of citrate 2
RMSD bond lengths (A
˚
) 0.011

RMSD bond angles (°) 1.5
Average B-factors (A
˚
2
)
Protein 29.5
NAD 26.9
Citrate 34.2
Water 33.4
a
Hg1, ethyl mercuric phosphate.
b
Hg2, 1,4-diacetoxymercuri-2,3-dimethoxybutane.
c
Hg3, phenylmercury acetate.
d
Values in parentheses
are for the last resolution shell.
e
R
sym
¼ S
h
S
i
| I
i
(h) –<I (h) >|⁄S
h
S

i
| I
i
(h) |,where I
i
(h) is the intensity measurement for a reflection h
and < I (h) > is the mean intensity for this reflection.
f
R
cryst
¼ S
h
jjF
obs
j–jF
calc
jj ⁄S
h
jF
obs
j
.
g
The R
free
was calculated with randomly selected
reflections (10%).
K. Yoneda et al. Crystal structure of
L-aspDH from A. fulgidus
FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4317

are within hydrogen-bonding distance of the side chain
of K134, the main chain amide proton of L161, and a
water molecule (WAT33). All of the residues are con-
served in T. maritima l-aspDH, except for L161, which
is replaced with Ile. Within the active site of the
T. maritima enzyme, however, we found that the side
chains of residues corresponding to K134 in loop 1,
N187 in loop 2 and S216 in loop 3 are far removed
from the l-aspartate molecule, and it does not appear
that hydrogen bonds are formed with the carboxyl
groups of the substrate (Figs 3A and 4B). This
suggests that the large movement of loops 1 and 2, in
addition to the formation of a flap by loop 3, may
induce the suitable positioning of the three residues
(K134, N187 and S216) for substrate binding and the
expression of l-aspDH’s catalytic activity.
In general, NAD(P)H-dependent dehydrogenases
show pro-R or pro-S stereospecificity for hydrogen
removal from the C4 position of the nicotinamide moi-
ety of NAD(P)H. In our binding model, the re-face of
the nicotinamide ring is in front of the a-hydrogen
atom of substrate (Fig. 4B), and that is in good agree-
ment with our earlier finding that A. fulgidus l-aspDH
belongs to the dehydrogenase group with pro-R-spe-
cific hydrogen transfer [5].
Cofactor binding
The electron density corresponding to the NAD coen-
zyme bound within the active site was very clear,
which enabled us to place the ligand with reasonable
accuracy (Fig. S1). The map enabled clear positioning

of the adenine ring, identification of the C2¢-endo
A
B
Fig. 1. (A) The A. fulgidus L-aspDH dimeric model viewed down the
twofold noncrystallographic rotation axis. The A and B subunits are
shown in rainbow and gray, respectively. (B) Ribbon plot of the
A. fulgidus
L-aspDH monomer. The NAD binding and catalytic
domains are shown in green and blue, respectively. NAD (magenta)
and citrate (yellow) are shown as stick models.
Fig. 2. Structure-based amino acid sequence alignment of A. fulgi-
dus and T. maritima
L-aspDHs (AF, Archaeoglobus fulgidus; TM,
Thermotoga maritima). Sequences were aligned using
CLUSTAL W
[35]. Boxes represent conserved residues in the enzymes. The resi-
dues that are disordered in T. maritima
L-aspDH are shown in pur-
ple. The residues involved in citrate and NAD binding are shown in
blue and red, respectively. The secondary structural assignments
for the A. fulgidus
L-aspDH structure are shown above the align-
ment in green; those for the T. maritima
L-aspDH structure are
shown below the alignment in blue.
Crystal structure of
L-aspDH from A. fulgidus K. Yoneda et al.
4318 FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS
conformation of the two ribose rings, clear definition
of the backbone phosphate groups and assignment of

an anti conformation to the glycosidic bond linking
the nicotinamide ring and its associated ribose moiety.
N7, N1 and N6 of the adenine base, respectively,
formed hydrogen bonds with the side chains of S59
and Y66 and, via a water molecule (WAT26), with the
side chain of D65. The hydroxyl groups of the adenine
ribose interact with R33 using side-on geometry. A
glycine-rich motif, GXGXXG, which lies close to the
adenine ribose, dictates the nature of the hydrogen
bonding between the main chain and the adenine
ribose moiety [7]. In A. fulgidus l-aspDH, this motif is
recognized at the position of the 7–12th amino acids
from the N-terminus (Fig. 2). It is known that the
occurrence of an aspartic or glutamic acid residue at
the C- or N-terminus of the second b strand of the bab
fold is a common feature of NAD(P)-dependent dehy-
drogenases [8–12]. The acidic residue plays an impor-
tant role in the formation of hydrogen bonds with the
adenine ribose hydroxyl groups of the cofactor [13,14].
In A. fulgidus l-aspDH, D31, which is at the N-termi-
nal end of b2, forms hydrogen bonds with the 2¢ and
3¢ hydroxyl groups of the adenine ribose of NAD
(Fig. 5). The adenine phosphate interacts with A10,
I11, a water molecule (WAT119) and, via water mole-
cules, with R33 (WAT125) and A57 (WAT5). In addi-
tion, the nicotinamide phosphate interacts with N212,
T215 and a water molecule (WAT119) and, via a water
molecule (WAT5), with A57. The hydroxyl groups of
the nicotinamide ribose interact with the side chain of
S81, N162 and backbone oxygen of A58. The hydro-

gen-bonding pattern between the enzyme and NAD is
completed by interactions between the carboxyamide
moiety of the nicotinamide ring and the main-chain
NH of A111, the main-chain oxygen of L80, the side
chain of T166 (via water molecule WAT38), and the
main-chain oxygen of A108. These hydrogen bonds
lead to an anti conformer for the glycosidic bond
between the nicotinamide ring and its associated
ribose, and enable the 4-pro-R hydrogen atom to be
involved in the hydride-transfer step.
Accessible surface area and hydrogen bonds
It is thought that, in general, a reduction in the sol-
vent-accessible surface area and an increase in the frac-
tion of buried hydrophobic atoms are the stabilizing
principles that serve as the basis for protein thermo-
stability [15]. As shown in Table 2, the total solvent-
accessible surface area of A. fulgidus l-aspDH
(monomer, 12 665 A
˚
2
; dimer, 20 685 A
˚
2
) is similar to
that of T. maritima l-aspDH (monomer, 12 892 A
˚
2
;
A
B

Fig. 3. Comparison of the structures of A. fulgidus L-aspDH and
T. maritima
L-aspDHs. (A) The superimposed Ca-traces of A. fulgi-
dus
L-aspDH and T. maritima L-aspDH; the structures of the two
enzymes are shown in green and magenta, respectively. NAD
(magenta) and citrate (yellow) molecules are shown as sphere mod-
els. (B) Scheme around the substrate binding loop in the catalytic
domain. The citrate molecule is shown as a stick model in yellow.
Oxygen and nitrogen atoms are shown in red and blue, respec-
tively.
L-AspDHs are colored as in (A).
K. Yoneda et al. Crystal structure of
L-aspDH from A. fulgidus
FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4319
dimer, 21 381 A
˚
2
). Likewise, the total interface area
between A. fulgidus l-aspDH subunits A–B (2322 A
˚
2
)
is similar to that between the T. maritima l-aspDH
subunits (2201 A
˚
2
), and the total hydrophobic areas
of the interfaces are about the same (972–973 A
˚

2
)
(Table 2). There was also no significant difference in
the number of hydrophobic residues (108 and 113 resi-
dues in A. fulgidus and T. maritima l-aspDHs, respec-
tively) between the two enzymes. Using insight ii
(Biosym ⁄ MSI), the number of hydrogen bonds within
A
B
Fig. 4. Stereoview of the citrate coordinated
within the active site and the proposed bind-
ing model for the
L-aspartate molecule.
(A) Schematic representation depicting the
interactions between citrate and the protein.
The networks of hydrogen bonds are shown
by dotted lines. The citrate molecule is
shown as a stick model in yellow. The final
r
A
-weighted 2|F
o
| ) |F
c
| electron-density
map for citrate is shown at the 1r level.
(B) Comparison of the A. fulgidus
L-aspDH
active site pocket with that of T. maritima
L-aspDH and the proposed binding model of

the
L-aspartate. The C4 atom of the pyridine
ring (a hydride acceptor site), and si- and
re-faces are labeled. The
L-aspartate mole-
cule is shown as a stick model in cyan. The
structures of A. fulgidus and T. maritima
L-aspDH are shown in green and white,
respectively. Atoms are colored as
described for Fig. 3.
Fig. 5. Stereo representation of NAD bound
to A. fulgidus
L-aspDH. Residues that inter-
act with NAD are labeled. The networks of
hydrogen bonds are shown as dotted lines.
Atoms are colored as described for Fig. 3.
Crystal structure of
L-aspDH from A. fulgidus K. Yoneda et al.
4320 FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS
the A. fulgidus and T. maritima l-aspDH monomers
were determined to be 149 and 131, respectively, and
the number of intersubunit hydrogen bonds was 26
and 28, respectively. Again, the two enzymes showed
considerable similarity.
Comparison of the amino acid compositions
The higher numbers of Pro at the N1 position or Ala
and lower numbers of b-branched residues (Val, Thr
and Ile) are known to correlate significantly with the
thermostability of proteins by stabilizing their a helices
[16–18]. The total numbers of Pro at the N1 position

and b-branched residues were 0 and 15, respectively, in
the a helices of A. fulgidus l-aspDH, and 1 and 18,
respectively, in the T. maritima enzyme. By contrast,
the total numbers of Ala residues in the a helices
of A. fulgidus and T. maritima l-aspDHs were 15
and 5, respectively. Thus, A. fulgidus and T. maritima
l-aspDHs do not differ significantly with respect to
their Pro or b-branched residue contents; however, the
relatively high Ala content in the a helices of the A. ful-
gidus l-aspDH might contribute to its thermostability.
Ion-pair interaction
Recent studies of the structures of hyperthermophilic
proteins have shown that the number of ion pairs and
the formation of ion networks contribute significantly
to the thermostability of these enzymes [19–21]. We
examined the structural characteristics of the A. fulgi-
dus [5] and T. maritima l-aspDHs from the aspect
of high thermostability. The stability of T. maritima
l-aspDH has not been reported to date. Thus, we
expressed the enzyme gene in E. coli and purified the
recombinant enzyme to homogeneity. Thermostability
of the enzyme was then compared with that of A. ful-
gidus l-aspDH. The half-life of A. fulgidus l-aspDH
(t
1 ⁄ 2
¼ 10.0 min) was similar to that of T. maritima
enzyme (t
1 ⁄ 2
¼ 10.7 min) at 100 °C, indicating that the
thermostabilities of the two enzymes are comparable.

Using a cut-off distance of 4.0 A
˚
between oppositely
charged residues, we calculated that A. fulgidus
l-aspDH contained 16 intrasubunit ion pairs, whereas
T. maritima l-aspDH contained only nine (Table 2).
In addition, three major intersubunit ion-pair interac-
tions were observed in A. fulgidus l-aspDH: E201–
R203¢, R203–E201¢ and R95–E232¢ (the prime indi-
cates the neighboring subunit in the dimer). An ion
pair between E232 and R95¢ could not be observed
because of the poor electron density of the side chains.
Four (E201, R203, E201¢, and R203¢) of the residues
are located within b11 and b11¢, and form a four-resi-
due ion-pair network between the A and B subunits
(Fig. 6). In the T. maritima l-aspDH, the charged R95
is replaced by aromatic F93, which is involved in the
largest aromatic pair network in T. maritima enzyme
(see below). In addition, T. maritima l-aspDH con-
tains no ion-pair networks (Table 2).
Aromatic pair interaction
Aromatic interactions are also known to participate in
stabilizing protein structure [22,23]. A pair of aromatic
Table 2. Comparison of ion-pair interactions, aromatic pair interac-
tions, accessible surface areas, and hydrogen bonding in A. fulgidus
and T. maritima
L-aspDHs.
A. fulgidus T. maritima
PDB code 2DC1 1J5P
Resolution (A

˚
) 1.9 1.9
No. intrasubunit ion pairs
per subunit
16 9
No. intersubunit ion pairs 3 0
Ion pair network 1 · 4
residues
not observed
No. intrasubunit aromatic
pairs per subunit
611
No. intersubunit aromatic pairs 0 5
Largest aromatic pair network 2 · 4
residues
2 · 7 residues
Solvent-accessible surface area (A
˚
2
)
Monomer 12665 12892
Dimer 20685 21381
Interface 2322 2201
Hydrophobic interface 973 972
No. intrasubunit H bond
per subunit
149 131
No. intersubunit H bond 26 28
Fig. 6. The intersubunit ion pair network in A. fulgidus L-aspDH.
Residues belonging to the A and B subunits are shown in green

and magenta, respectively. Atoms are colored as described for
Fig. 3.
K. Yoneda et al. Crystal structure of
L-aspDH from A. fulgidus
FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4321
interactions contributes between )0.6 and )1.3 kcalÆ
mol
)1
to protein stability [24]. Using a cut-off distance
between the aromatic ring centers of 7.0 A
˚
, we deter-
mined that there are six intrasubunit aromatic pairs in
A. fulgidus l-aspDH (Table 2). By contrast, there are
11 aromatic pairs in T. maritima l-aspDH. The most
extensive aromatic pair network (seven residues; Y75,
F83, F88, F92, F93, F105, and F240¢) is located in the
Rossmann-fold domain (Fig. S2), and two of those
residues (F105 and F240¢) form an intersubunit aro-
matic pair. By contrast, the largest aromatic pair net-
work identified in A. fulgidus l-aspDH is composed of
only four residues (F14, W18, F24, and Y217) and is
located on the surface of the Rossmann-fold domain
(Fig. S2). It is noteworthy that whereas five major
intersubunit aromatic pair interactions (F105–F240¢,
F122–Y225¢, F206–F206¢, Y225–F122¢, and F240–
F105¢) were observed in T. maritima l-aspDH, there
are no intersubunit aromatic pairs in the A. fulgidus
enzyme (Fig. S2). Structure-based sequence alignment
showed that with the exception of F83 and F88, the

residues forming the largest aromatic pair network in
T. maritima l-aspDH were replaced by nonaromatic
residues in A. fulgidus l-aspDH (Fig. 7). Thus, while it
appears that intersubunit ion-pair interactions are the
primary mediators of the high thermostability of
A. fulgidus l-aspDH, the relatively large number of
intersubunit aromatic pairs suggests they are the major
mediators of T. maritima l-aspDH thermostability.
Experimental procedures
Protein expression and purification
Expression of the gene encoding A. fulgidus l-aspDH in
E. coli and its purification from the recombinant cells was
carried out as described previously [5].
Crystallization
For the crystallization trails, purified A. fulgidus l-aspDH
was dialyzed against 10 mm potassium phosphate
buffer (pH 7.2) containing 0.2 m NaCl. Crystallization of
l-aspDH was accomplished using the sitting-drop vapor-
diffusion method, and the initial screening was carried out
using Crystal Screen Cryo (Hampton Research, Aliso Viejo,
CA, USA) at 20 °C. The crystal obtained belonged to the
orthorhombic space group P2
1
2
1
2, and the unit cell param-
eters were a ¼ 47.52 A
˚
, b ¼ 89.58 A
˚

, c ¼ 100.49 A
˚
and
a ¼ b ¼ c ¼ 90°. The crystals were grown in sitting drops
in which 1 lL of enzyme solution (14.5 mgÆmL
)1
) contain-
ing 1 mm NAD was mixed with 1 lL of mother liquor con-
taining 100 mm phosphate-citrate buffer pH 4.2 (60.5 mm
Na
2
HPO
4
, 39.5 mm citric acid), 5% (v ⁄ v) polyethylene gly-
col 3000 (PEG 3000), 10% (v ⁄ v) glycerol and 22% (v ⁄ v)
1.2-propanediol.
Data collection
Crystals were coated with a layer of viscous oil (Paratone-N)
and transferred into a stream of nitrogen gas for data col-
lection at 100 K. Diffraction data were collected at a reso-
lution of 1.9 A
˚
on beamline KEK-NW12 at the Photon
Factory (Tsukuba, Japan) using monochromatized radia-
tion at k ¼ 1.0 A
˚
and an ADSC Quantum 210 CCD detec-
tor (Area Detector Systems, San Diego, CA, USA). The
oscillation angle per image was set to 1°, and the data were
processed using HKL 2000 [25]. Heavy atom derivatives

were prepared by soaking the crystals for 24 h in mother
B
A
Fig. 7. The largest aromatic pair network, formed by seven resi-
dues in T. maritima
L-aspDH (A), and the equivalent positions in
A. fulgidus
L-aspDH (B). Residues belonging to the A and B sub-
units are shown in green and magenta, respectively. Atoms are
colored as described for Fig. 3.
Crystal structure of
L-aspDH from A. fulgidus K. Yoneda et al.
4322 FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS
liquor containing 0.5 mm ethyl mercuric phosphate, 1 mm
1,4-diacetoxymercuri-2,3-dimethoxybutane or 1 mm phenyl-
mercury acetate.
Phasing and refinement
The native, ethyl mercuric phosphate, 1,4-diacetoxymercuri-
2,3-dimethoxybutane, and phenylmercury acetate data sets
were used for phase calculation (Table 1), which was
accomplished by MIRAS using solve [26]. The MIRAS
map at 1.9 A
˚
was subjected to maximum-likelihood density
modification, followed by autotracing using resolve [27].
An initial model was built using xtal view [28], after which
several cycles of rigid-body refinement, positional refine-
ment and simulated annealing were performed at 1.9 A
˚
res-

olution using refmac [29] and cns [30]. The model was
adjusted in xtal view using both |F
o
| ) |F
c
| and 2|F
o
| ) |F
c
|
maps. NAD and citrate molecules were clearly visible in
both the r
A
-weighted |F
o
| ) |F
c
| and 2|F
o
| ) |F
c
| maps, and
these molecules were included in the latter part of the refine-
ment. The current model contains 472 residues (A 1–236
and B 1–236), 136 water molecules, two NAD and two
citrate molecules. The model geometry was analyzed using
procheck [31], and 91.2% of the nonglycine residues were
in the most favored region of the Ramachandran plot and
8.8% in the additionally allowed region. Molecular graphics
were created using pymol ( />The coordinates and structure factors of A. fulgidus l-aspDH

complexed with NAD and citrate have been deposited in
the Protein Data Bank with the accession code 2DC1.
Structural analysis and comparison
Ion-pair interactions within the two structures were identi-
fied using the WHAT IF web server [32] with a cut-off
distance ¼ 4.0 A
˚
[33] between oppositely charged residues.
Aromatic interactions were defined using a cut-off dis-
tance of 7.0 A
˚
between the aromatic ring centers [22].
Hydrogen bonds were identified using the program
insight ii (Biosym ⁄ MSI, San Diego, CA, USA). Hydro-
gen was added to the coordinates of the proteins and the
calculated hydrogen bonds (the distance between a calcu-
lated hydrogen position and model oxygen or nitrogen
atom was < 3.0 A
˚
, and the angle between the proton
donor and acceptor is > 120°) were measured using the
criterion of insight ii. The solvent accessible surface area
was calculated using the program grasp [34].
Thermal stability
To assess the thermostability, the A. fulgidus or T. maritima
l-aspDH (0.1 mgÆmL
)1
)in10mm potassium phosphate
buffer (pH 7.2) containing 0.2 m NaCl was incubated at
100 °C, after which the residual activity of the enzymes was

determined at appropriate intervals using a standard assay
as described previously [5].
Acknowledgements
Data collection was performed at the Photon Factory
(Tsukuba, Japan). We thank Drs K. Demura, N. Mats-
ugaki, N. Igarashi and S. Wakatsuki for their kind assis-
tance with the data collection. 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 and by a Grant-in-Aid for Scientific Research (C)
from the Japan Society for the Promotion of Science.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Representative portion of the final r
A
-
weighted 2|F
o
|–|F
c
| electron-density map of A. fulgi-
dus
L-aspDH with bound NAD contoured at the 1r
level (blue), together with a fitted model of NAD
shown as a stick model in magenta.
Fig. S2. The inter- and intrasubunit aromatic pairs in
T. maritima
L-aspDH (A) and A. fulgidus L-aspDH
(B). The regions of the intersubunit aromatic pair and
the largest aromatic pair network are indicated by a
Crystal structure of L-aspDH from A. fulgidus K. Yoneda et al.
4324 FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS
circle and a broken-line circle, respectively. Residues
belonging to A and B molecules are shown in green
and magenta, respectively.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
K. Yoneda et al. Crystal structure of L-aspDH from A. fulgidus
FEBS Journal 274 (2007) 4315–4325 ª 2007 The Authors Journal compilation ª 2007 FEBS 4325