First crystal structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase

Article abstract of DOI:10.1074/jbc.M109.084384

A gene encoding an L-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable L-lysine dehydrogenase yet described, with a half-life of 180 min at 100 °C. The product of the enzyme's catalytic activity is Δ¹-piperideine-6-carboxylate, which makes this enzyme an L-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the ∈-amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 A in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of L-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase.

Full text of DOI:10.1074/jbc.M109.084384

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 11, pp. 8444–8453, March 12, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
First Crystal Structure of L-Lysine 6-Dehydrogenase as an
□
S
NAD-dependent Amine Dehydrogenase*
Received for publication, November 12, 2009, and in revised form, January 6, 2010 Published, JBC Papers in Press, January 7, 2010, DOI 10.1074/jbc.M109.084384
Kazunari Yoneda‡, Junya Fukuda§, Haruhiko Sakuraba¶, and Toshihisa Ohshima§1
From the ‡Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto 869-1404, the §Institute of Genetic
Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812-8581, and the ¶Department of Applied Biological
Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
A gene encoding an L-lysine dehydrogenase was identified
in the hyperthermophilic archaeon Pyrococcus horikoshii.
The gene was overexpressed in Escherichia coli, and its prod-
uct was purified and characterized. The expressed enzyme is
the most thermostable L-lysine dehydrogenase yet described,
with a half-life of 180 min at 100 °C. The product of the
enzyme’s catalytic activity is ⌬1-piperideine-6-carboxylate,
which makes this enzyme an L-lysine 6-dehydrogenase (EC
1.4.1.18) that catalyzes the reductive deamination of the ⑀-
amino group and a type of NAD-dependent amine dehydro-
genase. The three-dimensional structure of the enzyme was
determined using the mercury-based multiple-wavelength
catalyzes the oxidative deamination of the ⑀-amino group of
L-lysine to form L-2-aminoadipate 6-semialdehyde, which in
turn nonenzymatically cyclizes to form ⌬1-piperideine-6-car-
boxylate (P6C) (1–3). The second is L-lysine 2-dehydrogenase
(EC 1.4.1.15), which catalyzes the oxidative deamination of the
␣-amino group of L-lysine in the same way other amino acid
dehydrogenases do (4). With respect to the latter, there has
been one report of an L-lysine 2-dehydrogenase catalyzing the
deamination of the ␣-amino group in humans (5), but because
the reaction product of this liver enzyme has not yet been iden-
tified, it is still unclear whether or not the enzyme actually cat-
alyzes the oxidative deamination of L-lysine at the ␣-amino
group. By contrast, LysDH has been identified in several micro-
organisms, and some of those enzymes have been character-
ized. The first known LysDH was found in Agrobacterium
tumefaciens, where it plays a key role in L-lysine metabolism (1).
Since then, this enzyme has been extensively characterized
(6–9) and utilized for assaying L-lysine (9).
˚
anomalous dispersion method at a resolution of 2.44 A in the
presence of NAD and sulfate ion. The asymmetric unit con-
sisted of two subunits, and a crystallographic 2-fold axis gen-
erated the functional dimer. Each monomer consisted of a
Rossmann fold domain and a C-terminal catalytic domain,
and the fold of the catalytic domain showed similarity to that
of saccharopine reductase. Notably, the structures of sub-
units A and B differed significantly. In subunit A, the active
site contained a sulfate ion that was not seen in subunit B.
Consequently, subunit A adopted a closed conformation,
whereas subunit B adopted an open one. In each subunit, one
NAD molecule was bound to the active site in an anti-confor-
mation, indicating that the enzyme makes use of pro-R-spe-
cific hydride transfer between the two hydrides at C-4 of
NADH (type A specificity). This is the first description of the
three-dimensional structure of L-lysine 6-dehydrogenase as
an NAD-dependent amine dehydrogenase.
Recently, the gene encoding LysDH in Geobacillus stearo-
thermophilus was cloned and expressed in Escherichia coli,
after which the primary structure of the enzyme was deter-
mined (2). Since then, ϳ17 different amino acid dehydroge-
nases have been identified in various organisms (4, 10). Some of
these enzymes are useful for enzymatic analysis of amino acids,
oxo acids, and special enzymes, as well as for stereospecific
synthesis of L-amino acids and their analogs (4, 10, 11). In con-
trast to the abundant information available for glutamate, ala-
nine, leucine, and phenylalanine dehydrogenases (4), informa-
tion about the structure and catalytic mechanism of lysine
dehydrogenase is still rather limited. LysDH is regarded as a
type of NAD(P)-dependent amine dehydrogenase because it
catalyzes the dehydrogenation at the ⑀-carbon with deamina-
tion, whereas other amino acid dehydrogenases generally cata-
lyze the oxidative deamination of the ␣-amino group to form
the corresponding keto acid. Although quinone- or heme-de-
pendent amine dehydrogenases have been extensively studied
L-Lysine dehydrogenase catalyzes the oxidative deamination
of L-lysine in the presence of NAD. To date, two types of L-ly-
sine dehydrogenase have been identified (Fig. 1). The first is
L-lysine 6-dehydrogenase (LysDH2; EC 1.4.1.18). This enzyme
* This work was supported in part by the “National Project on Protein Struc- (12–15), nothing is currently known about the structural basis
tural and Functional Analysis” promoted by the Ministry of Education, Sci-
for the catalytic mechanism of NAD(P)-dependent amine
ence, Sports, Culture, and Technology of Japan and by a grant-in-aid for
dehydrogenase.
scientific research (C) from the Japan Society for the Promotion of Science.
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. 1.
□S
Within the genomic sequence of an anaerobic hyperthermo-
philic archaeon, Pyrococcus horikoshii, we found a gene (open
reading frame identification number PH1688.1) whose pre-
dicted amino acid sequence exhibits 33% identity to that of
The atomic coordinates and structure factors (code 3ABI) have been depos-
ited in the Protein Data Bank, Research Collaboratory for Structural Bioin-
formatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence should be addressed. Tel.: 81-92-642-3053; Fax:
81-92-642-3059; E-mail: ohshima@agr.kyushu-u.ac.jp.
2 The abbreviations used are: LysDH, L-lysine 6-dehydrogenase; P6C, ⌬1-piperi-
deine-6-carboxylate; P2C, ⌬1-piperideine-2-carboxylate; BisTris, 2-[bis(2-hy-
droxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MAD, multiple-
wavelength anomalous dispersion.
8444 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of L-Lysine Dehydrogenase
an NAD(P)-dependent amine dehy-
drogenase, and it provides new
insight into the catalytic mechanism
of LysDH.
EXPERIMENTAL PROCEDURES
Cloning and Protein Expression—
The gene encoding LysDH (open
reading frame identification num-
ber PH1688.1; the gene information
is available at the DOGAN Genomic
Database) was amplified by PCR.
The oligonucleotide primers used
to amplify the LysDH gene fragment
were 5Ј-GAATCATATGAAAGT-
CCTTATTTTAGGG-3Ј, which
contains a unique NdeI restriction
site overlapping the 5Ј-initiation co-
don, and 5Ј-GTTAAGCTTTTAA-
GTTCCTTCTATAGATATTCC-
3Ј, which contains a unique HindIII
restriction site proximal to the
3Ј-end of the termination codon.
Chromosomal P. horikoshii DNA
was isolated as described previously
(16) and used as the template. The
amplified 1.0-kb fragment was
digested with NdeI and HindIII and
ligated with the expression vector
pColdI (Takara Bio Inc.) linearized
with NdeI and HindIII to generate
pCLysDH, which was then used to
transform E. coli strain Rosetta-
gami (DE3) (Novagen). The trans-
formants were cultivated at 37 °C in
2 liters of Luria-Bertani medium
until the absorbance at 600 nm
reached 0.5. Expression was then
induced by addition of 0.1 mM iso-
propyl ␤-D-thiogalactopyranoside
to the medium, and cultivation was
continued for an additional 24 h at
15 °C. P. horikoshii LysDH could be
easily obtained also using pET11a
or pET15b; however, the pColdI
FIGURE 1. Reaction scheme for the two types of L-lysine dehydrogenase. The proposed mechanism of the
P. horikoshii LysDH-catalyzed reaction is boxed.
express system was needed to
produce an enzyme suitable for
crystallization.
Site-directed Mutagenesis—Site-directed mutagenesis was
accomplished using a QuikChange XL site-directed mutagen-
esis kit (Stratagene) according to the manufacturer’s instruc-
tions. pCLysDH served as the template, and the following oli-
gonucleotide primers were used as the mutagenic primers (with
the mutations underlined): 5Ј-CTAATAGAGCAGTACACA-
G. stearothermophilus LysDH. This hyperthermostable LysDH
is a potentially useful target for the development of new meth-
ods for synthesis of L-pipecolic acid (chiral pharmaceutical
intermediate). Up to now, no LysDH has been reported in
Archaea, the third domain of life, or in hyperthermophiles. In
this study, we expressed the gene, characterized the enzyme
produced, and revealed that the enzyme is an extremely ther- AGGCCTGCT-3Ј and 5Ј-AGCAGGCCTTGTGTACTGCTC-
mostable LysDH. We also determined the crystal structure of TATTAG-3Ј for E168Q, 5Ј-CTAATAGAGGCGTACACAAG-
this enzyme at 2.44-Å resolution in the presence of NAD and GCCTGCT-3Ј and 5Ј-AGCAGGCCTTGTGTACGCCTCTA-
sulfate ion. This is the first description of the crystal structure of TTAG-3Ј for E168A, 5Ј-GGACGCTAAAGTGGCCTGGAC-
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Crystal Structure of L-Lysine Dehydrogenase
ATCTAG-3Ј and 5Ј-CTAGATGTCCAGGCCACTTTAGCG- ⌬1-piperideine-2-carboxylate (P2C) and P6C. As controls, P2C
TCC-3Ј for R226K, and 5Ј-CCCCTTTATTTTAAGATAACA- and P6C were therefore prepared by oxidation of L-lysine using
TGGTCCCCCAGGG-3Ј and 5Ј-CCCTGGGGGACCATGTT- L-lysine oxidase from Trichoderma viride (18) and by dehy-
ATCTTAAAATAAAGGGG-3Ј for Y156F.
drogenation of L-lysine using L-lysine dehydrogenase from
Purification from the Soluble Fraction—Cells were harvested G. stearothermophilus (2), respectively, as described previously.
by centrifugation, suspended in 20 mM Tris-HCl buffer (pH 7.5) The reaction mixture (1.0 ml) containing 10 mM L-lysine, 10 mM
containing 5 mM ␤-mercaptoethanol, and disrupted by sonica- NAD, 0.2 M Na2CO3 buffer (pH 9.5), and 1.6 mg of enzyme was
tion. The crude extract was heated at 80 °C for 10 min and then incubated at 70 °C for 15 min. After addition of 0.2 ml of 10%
clarified by centrifugation (10,000 ϫ g for 20 min), after which trichloroacetic acid and centrifugation, the supernatant solu-
the protein in the supernatant was run on a Ni2ϩ-chelating tion (0.5 ml) was run on a column (4.6 ϫ 120 mm) of TSKgel
Sepharose column (Amersham Biosciences) equilibrated Aminopak (Tosoh); citrate buffer (0.2 M, pH 3.25) served as the
with 20 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl, mobile phase at a flow rate of 0.4 ml/min at 55 °C. The eluate
0.1 M imidazole, and 5 mM ␤-mercaptoethanol. After wash- was collected in 1.2-ml portions and mixed with 0.2 ml of 50
ing the column with the same buffer, the enzyme was eluted mM ␱-aminobenzaldehyde in 0.2 M potassium phosphate buffer
with 0.5 M imidazole in the same buffer; the active fractions (pH 8.0). The absorbance was then measured at 460 nm after
were pooled; and the enzyme solution was dialyzed against incubation at 37 °C for 2 h.
20 mM Tris-HCl (pH 7.5). Purified P. horikoshii LysDH was
concentrated using an Amicon Ultra-15 unit (Millipore) and mine the effect of temperature on its stability, the enzyme was
used for crystallization. incubated at different temperatures in 20 mM Tris-HCl buffer
Stability, pH Optimum, and Kinetic Parameters—To deter-
Purification from Inclusion Bodies—To purify LysDH from (pH 7.5) containing 5 mM ␤-mercaptoethanol. The residual
inclusion bodies, E. coli transformants were collected, sus- activity was determined at appropriate intervals using the
pended in 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM standard assay method. To determine the effect of pH on its
␤-mercaptoethanol and 4% Triton X-100, and disrupted by stability, the enzyme in buffer at various pH values was incu-
sonication. After sonication, the suspension was centri- bated at 50 °C for 30 min, and the remaining activity was again
fuged, and the pellet was solubilized in 50 mM Tris-HCl determined using the standard assay method. The buffers (250
buffer (pH 7.5) containing 5 mM ␤-mercaptoethanol, 6 M mM) used for these assays were acetate (pH 4.5–6.0), BisTris-
guanidine HCl, 0.2 M NaCl, and 1 mM EDTA. Once solubi- HCl (pH 5.5–7.5), Tris-HCl (pH 7.5–9.0), glycine-KOH (pH
lized, LysDH was added to refolding buffer containing 0.1 M 8.5–11.0), and Na2HPO4-NaOH (pH 10.5–12.0). The same
Tris-HCl (pH 7.5), 5 mM ␤-mercaptoethanol, 0.4 M arginine, buffers were also used to determine the optimal pH for enzyme
2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride and activity at 70 °C. The initial velocity was determined by varying
incubated for 36 h at 4 °C. Refolded LysDH was heated at the concentration of one substrate while keeping the concen-
80 °C for 10 min and clarified by centrifugation, after which trations of the other substrates fixed as described previously
the protein in the supernatant was run on a Superdex 200 (19).
column (Amersham Biosciences) equilibrated with 20 mM
Crystallization and Data Collection—The initial screening
Tris-HCl buffer (pH 7.5) containing 5 mM ␤-mercaptoetha- for crystallization of LysDH was carried out with Wizard 1,2
nol and 200 mM NaCl, and the resultant protein solution was (Emerald BioStructures) at 20 °C using the sitting-drop
used for enzymological characterization.
vapor diffusion method. The crystal belongs to the ortho-
Determination of Enzyme Activity—Enzyme activity was rhombic space group P212121 with unit cell parameters a ϭ
assayed spectrophotometrically using a Shimadzu UV-1600 75.291, b ϭ 113.011, and c ϭ 122.456 Å and ␣ ϭ ␤ ϭ ␥ ϭ 90°.
spectrophotometer equipped with a thermostat. The standard The crystals were grown in sitting drops composed of 1 ␮l of
reaction mixture for oxidative deamination consisted of 100 enzyme solution (10 mg/ml) containing 1 mM NAD and 1 ␮l
mM glycine-KOH buffer (pH 10.0) containing 10 mM L-lysine, of mother liquor containing 100 mM citrate buffer (pH 5.8)
1.0 mM NAD, and the enzyme in a final volume of 1.0 ml. The and 2.1 M ammonium sulfate. Data were collected using an
pH values of buffer and lysine solutions were adjusted at room ADSC CCD detector system on beamline BL5A at the Pho-
temperature and used for the assay without temperature com- ton Factory in Tsukuba, Japan. All diffraction measurements
pensation. After warming the reaction mixture by incubation were carried out on crystals cryoprotected with 30% (v/v)
for 3 min at 70 °C without the coenzyme, the reaction was glycerol and cooled to 100 K in a stream of nitrogen gas. The
started by addition of the coenzyme. The appearance of NADH native and derivative data were processed using HKL2000
was monitored from the absorbance at 340 nm (extinction coef- (20).
ficient (⑀) of 6.22 mMϪ1 cmϪ1). One unit of activity is defined as
Phasing, Refinement, and Structure Analysis—Heavy atom
the amount of enzyme producing 1.0 ␮mol of NADH/min at derivatives were prepared by soaking the crystals in reservoir
70 °C under the standard assay conditions, and the specific solution containing 1 mM thimerosal for 22 h. Phase calcu-
activity is expressed in units/mg of protein. The protein con- lation was carried out using the multiple-wavelength anom-
centration was determined using the Bradford method (17); alous dispersion (MAD) method with SOLVE (21). The
bovine serum albumin served as the standard.
MAD map at 2.57 Å was subjected to maximum-likelihood
Identification of the Product—The product of the reaction density modification, followed by autotracing using RESOLVE
catalyzed by LysDH was analyzed by high performance liquid (22). The resultant model was built using XtalView (23) and
chromatography. Two possible products were postulated: refined against the native data set at 2.44-Å resolution using
8446 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of L-Lysine Dehydrogenase
FIGURE 2. A, effect of temperature on LysDH activity. Enzyme activity was measured at various temperatures between 50 and 95 °C. B, effect of temperature on
the stability of LysDH. The enzyme was incubated at 100 °C, and the residual activity was measured at the indicated times. C, effect of pH on LysDH activity.
Enzyme activity was measured at pH values ranging from 7.8 and 10.7. D, effect of pH on the stability of LysDH. The enzyme was incubated at 50 °C for 30 min
in buffers at various pH values, after which residual activity was measured.
RESULTS AND DISCUSSION
Refmac5 (24) and CNS (25). The NAD molecule and sulfate
ion were clearly visible in both the ␴A-weighted 2Fo Ϫ Fc and
Expression of the Gene and Purification of the Recombinant
Fo Ϫ Fc density maps and were included in the latter part of
Enzyme—Within the sequence of the P. horikoshii genome
the refinement. The R factor and Rfree values for the final
(DOGAN Genomic Database), we identified a gene, PH1688.1
model were 22.45 and 23.55%, respectively (Table 1). The
(1050 bp at positions 1486245–1485196 of the genome), whose
current model contains 694 amino acid residues (subunit A,
predicted amino acid sequence shows 33% identity to that of
0–348; and subunit B, 0–157 and 162–348), 135 water mol-
G. stearothermophilus LysDH. After transforming E. coli using
ecules, two NAD molecules, and two sulfate ions. The model
an expression vector harboring this gene (pCLysDH), the trans-
formant cells exhibited LysDH activity, and the enzyme was
readily purified from their crude extract in two simple steps:
heat treatment and affinity chromatography. Only 1.7 mg of the
purified enzyme was obtained from 7.3 g of cells (wet weight),
however; and the pattern on SDS-PAGE showed that P. horiko-
shii LysDH was present mainly as an inclusion body. For pro-
tein purification from the inclusion body, the collected precip-
geometry was analyzed using PROCHECK (26); 94.5% of the
non-glycine residues were in the most favored region of the
Ramachandran plot, and 5.1% were in the additionally
allowed region. In both subunits, Arg209 was found in a dis-
allowed conformation, which may be explained by its loca-
tion in a sharp turn between ␣10 and ␤11. Hydrogen bonds
were identified using the program CCP4mg (27, 28). Molec-
ular graphics were created using PyMOL.
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Crystal Structure of L-Lysine Dehydrogenase
TABLE 1
Statistics of data collection, phase determination, and refinement
FOM, figure of merit; r.m.s.d., root mean square deviation.
Native
Peak
Edge
Remote
Data collection
Wavelength (Å)
1.0000
0.9932
2.57
1.0090
2.57
0.99284
2.57
Maximum resolution (Å) 2.44
No. of unique reflections
Redundancy
39,442
63,980
63,926
3.8
63,960
3.7
7.1
3.7
Completeness (%)a
99.6 (96.3)
5.9 (26.4)
15.5 (5.0)
99.6 (96.7)
6.6 (29.2)
14.9 (4.2)
0.47 (84.5-2.57)
99.7 (97.4) 99.4 (95.4)
5.2 (22.7) 6.3 (30.5)
15.2 (4.2) 14.6 (3.8)
R
sym (%)a,b
a
͗I/␴(I)͘
MAD phasing FOM
Refinement
Resolution range (Å)
47.5–2.44
22.45
23.55
5563
135
R
R
cryst (%)c
free (%)d
No. of protein atoms
No. of water molecules
No. of NAD molecules
No. of sulfate ions
2
2
r.m.s.d. bond length (Å)
r.m.s.d. bond angle
0.010
1.6°
a Values in parentheses are for the last resolution shell.
b Rsym ϭ ⌺h⌺i͉Ii(h) Ϫ ͗I(h)͉͘/⌺h⌺i͉Ii(h)͉, where Ii(h) is the intensity measurement for
reflection h and ͗I(h)͘ is the mean intensity for this reflection.
c Rcryst ϭ ⌺hʈFo͉ Ϫ ͉Fcʈ/⌺h͉Fo͉.
d Rfree was calculated with randomly selected reflections (10%).
FIGURE 3. A, ion-exchange chromatography of the reaction product formed
from L-lysine; B, authentic P2C and P6C.
itate was dissolved in a 6 M guanidine HCl solution with gentle
stirring, after which the dissolved protein was added to a refold-
ing buffer (see “Experimental Procedures”). Once refolded,
P. horikoshii LysDH was further purified by heat treatment and
gel filtration chromatography. This procedure increased our
yield to ϳ7.8 mg of the purified enzyme from 7.3 g of cells (wet
weight). The specific activities of soluble and refolded LysDHs
at 70 °C were estimated to be ϳ0.31 and 0.45 units/mg, respec-
tively. At 70 °C, the specific activity of P. horikoshii LysDH was
rather less than that of G. stearothermophilus (6.2 units/mg at
50 °C) or A. tumefaciens LysDHs (4.54 units/mg at 37 °C), but as
FIGURE 4. A, overall structure of P. horikoshii LysDH. The NAD-binding and
catalytic domains are shown in cyan and pink, respectively. The adjacent sub-
unit is shown in gray. The region involved in dimer formation is indicated.
B, ribbon plot of the P. horikoshii LysDH monomer. NAD (magenta) and the
sulfateion(yellow)areshownasstickmodels. Oxygenandnitrogenatomsare
shown in red and blue, respectively.
shown in Fig. 2A, the activity of P. horikoshii LysDH dramati- enzyme corresponded to the predicted sequence, including the
cally increased with increases in the assay temperature, and the histidine tag.
value at 95 °C was 11 times greater than that at 70 °C. Indeed,
Molecular and Catalytic Properties—SDS-PAGE showed the
still greater activity would likely have been observed at the opti- subunit molecular mass of P. horikoshii LysDH to be ϳ42.6
mum growth temperature (ϳ104 °C) of this hyperthermophile. kDa, which was consistent with the molecular mass (42,089 Da)
The purified enzyme showed a single protein band upon SDS- calculated from the amino acid sequence. That the native
PAGE, and the N-terminal amino acid sequence of the purified molecular mass determined by gel filtration was ϳ97.2 kDa
8448 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of L-Lysine Dehydrogenase
L-lysine, which would be advanta-
geous for enzymatic detection of
L-lysine or for use as an L-lysine
biosensor.
Both NAD and NADP were able
to serve as the coenzyme for P. hori-
koshii LysDH, although the reaction
rate with NADP (1.0 mM) was only
7.8% of that with NAD (1.0 mM)
when L-lysine was the substrate.
The A. tumefaciens enzyme is
highly specific for NAD (8), whereas
the G. stearothermophilus enzyme
can utilize both NAD and NADP
(2). In this regard, P. horikoshii
LysDH is similar to the G. stearo-
thermophilus enzyme. We also
examined the effect of various
chemicals on the enzyme activity.
P. horikoshii LysDH was strongly
inhibited by HgCl2 (relative activity
with the additive was 17.3% of that
without the additive) and ZnCl2
(34.7%) but was unaffected by
EDTA, MgCl2, CaCl2, NiCl2, CoCl2,
CuCl2, SrCl2, or AlCl3 (each at a
concentration of 1 mM). P. horiko-
FIGURE 5. Active site of P. horikoshii LysDH and comparative analysis of subunits A and B. A, schematic
shii LysDH showed typical Michae-
lis-Menten kinetics, and the Km val-
ues for NAD and L-lysine were 0.080
and 1.17 mM, respectively.
representation depicting the interactions between the sulfate ion and the protein in subunit A. The networks
of hydrogen bonds are shown as dashed lines. The sulfate ion is shown as a stick model in yellow. The final
␴A-weighted 2Fo Ϫ Fc electron density map for the sulfate ion and the active site in subunit A is shown at the 1␴
level. The C-4 atom of the pyridine ring (a hydride acceptor site) and the si- and re-faces are labeled. B, surface
representation of subunits A (magenta) and B (yellow).
Identification of the Reaction
Product—The product of the reac-
suggests that native LysDH is a homodimer. When we tested tion catalyzed by P. horikoshii LysDH was identified using high
the effect of temperature on enzyme activity, we observed max- performance liquid chromatography. The reaction product
imum activity at ϳ95 °C and an extraordinary drop off in activ- from L-lysine was separated as the same peak as authentic P6C
ity with reduction in temperature; the activity at 50 °C was only (Fig. 3). In addition, orange-colored complexes of the reaction
ϳ1/170th of that at 95 °C (Fig. 2A). On the other hand, the product with ␱-aminobenzaldehyde showed the same absorp-
enzyme was highly thermostable and retained full activity even tion spectrum as ␱-aminobenzaldehyde in complex with
after incubation for 50 min at 100 °C (pH 7.5). Indeed, the half- authentic P6C, which had an absorption maximum at 460 nm.
life of the enzyme at 100 °C was 180 min (Fig. 2B and supple- This was clearly different from the spectrum of ␱-aminobenz-
mental Fig. 1), making P. horikoshii LysDH the most thermo- aldehyde in complex with authentic P2C, which had an absorp-
stable LysDH described to date.
tion maximum at 445 nm (1). Thus, the ⑀-amino group of
The enzyme was most active at alkaline pH, with the highest L-lysine is oxidatively deaminated by the enzyme, yielding L-2-
activity at pH ϳ10, as is the case with other amino acid dehy- aminoadipate 6-semialdehyde, which spontaneously converts
drogenases (Fig. 2C). Nonetheless, it was stable over a wide to the dehydrated form, P6C (Fig. 1).
range of pH values, losing no activity when incubated at pH
These results indicate that the gene PH1688.1 encodes a
LysDH, making this the first identification of this enzyme in
values between 5.0 and 12 for 30 min at 50 °C (Fig. 2D).
Examination of the substrate specificity of P. horikoshii Archaea. When we investigated the structure of PH1688.1 in
LysDH revealed that L-lysine is the preferred substrate for oxi- the data base, we found that the gene did not cluster with any
dative deamination in the presence of NAD, although the other genes, and no similar gene was found in other strains of
enzyme showed some activity with S-(␤-aminoethyl)-L-cys- Pyrococcales, suggesting that LysDH may play a specific role
teine. (With 10 mM substrate, the relative activity was 10.6% of during lysine metabolism in P. horikoshii.
that seen with L-lysine.) D-Lysine, L-ornithine, L-saccharopine,
Overall Structure—The structure of P. horikoshii LysDH was
⑀-amino-n-capronate, ␣-aminoadipate, ⌬-aminovalerate, L-al- determined using mercury-based MAD and was refined at a
anine, L-glutamine, L-asparagine, L-arginine, L-glutamate, and resolution of 2.44 Å (Table 1). The asymmetric unit consisted of
L-aspartate were inert. Like A. tumefaciens and G. stearother- one homodimer with a solvent content of 59.5%, which corre-
mophilus LysDHs, P. horikoshii LysDH is highly specific for sponds to a Matthews coefficient (29) of 3.1 Å3/Da. The model
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Crystal Structure of L-Lysine Dehydrogenase
of dimeric LysDH consisted of 694 amino acid residues, two
NAD molecules, two sulfate ions, and 135 water molecules. The
three-dimensional structure of the N-terminal histidine tag
Ϫ15
Ϫ1
region (Met
to Arg in both subunits), the C-terminal
region (Thr349 in both subunits), and the substrate-binding
loop (Ile158–Ser161 in subunit B) could not be modeled in this
study because of poor electron density. Well defined electron
density for Pro120 and Pro153 indicated a cis-conformation for
the peptide bond. The two subunits had approximate dimen-
sions of 49 ϫ 37 ϫ 52 Å and were related by a 2-fold non-
crystallographic rotation axis (Fig. 4A). Each monomer con-
sisted of two domains: an N-terminal coenzyme-binding
domain (␣1–␣7, ␣14, ␤1–␤6, ␤15), which formed a classical
Rossmann fold motif, and a C-terminal domain (␣8–␣13, ␣15-
␣16, ␤7–␤14, ␤16), within which both the catalytic activity and
formation of the homodimer were mediated (Fig. 4B). The
dimer associated through hydrophobic interactions between
␣11, ␣12, and loop 147–157 of both subunits, which formed a
stable four-helix bundle (Fig. 4A). The surface area buried in the
dimer interface was 1369 Å2 (calculated using a probe with a
radius of 1.4 Å; Protein-Protein Interface Analysis Server),
which is 4.5% of the total surface area of the enzyme. Multiple
oligomeric structures, including hexameric, dimeric, and tet-
rameric forms, have been observed for LysDHs from bacteria.
The enzyme from G. stearothermophilus adopts a hexameric
structure (2), whereas that from A. tumefaciens reportedly
forms a dimeric or tetrameric structure and binds 2 mol of
NADH/mol of tetrameric enzyme (the dimeric enzyme has one
catalytic site) (30). In this regard, P. horikoshii LysDH differs
substantially from these two previously described LysDHs.
Structural Differences between the Two Subunits—In the
electron density map of P. horikoshii LysDH obtained from our
preliminary experimental data, we noticed an extra density
within the active-site cavity in subunit A and found that a sul-
fate ion could be modeled into that density after construction
and refinement of the peptide chain. The map clearly defined
the precise orientation of the sulfate ion (Fig. 5A): its two oxy-
gen atoms formed hydrogen bonds with the side chains of
Trp160 and Arg226 and a proton in the main chain amide of
Gly145. In subunit B, however, we found no sulfate ion in the
active site; instead, it was on the surface of the Rossmann fold
domain. Moreover, according to calculations carried out using
the DynDom program for protein domain motion analysis (31–
33), the Rossmann fold domain in subunit A was rotated 11.2°
FIGURE 6. A, comparison of the structures of P. horikoshii LysDH and M. grisea
saccharopine reductase. The structure of P. horikoshii LysDH is shown in yel-
low; that of M. grisea saccharopine reductase is shown in green (Domain I),
cyan (Domain II), and magenta (Domain III). B, overall structure of M. grisea
saccharopine reductase. The structure is colored as described for Fig. 3. The
region involved in dimer formation is indicated.
relative to that in subunit B, which leads to 99% active-site clo- proteins showed structural similarity to LysDH, although these
sure in subunit A (Fig. 5B). Structural comparison between cases are not discussed here in more detail. With regard to the
LysDH subunits A and B showed that whereas the former C-terminal domain, the protein with the highest structural sim-
assumes a closed conformation, the latter assumes an open one ilarity was the saccharopine reductase from Magnaporthe
(root mean square deviation of C-␣ positions between A and grisea (Protein Data Bank code 1FF9) (35), which had a Z-score
B ϭ 1.31 Å). Within the ternary complex, the sulfate in the of 15.1 and a root mean square deviation of 2.7 Å over 198 C-␣
active site of subunit A occupies a position that corresponds to positions. The saccharopine reductase folds into three
the Lys carboxylate-binding site in our model (Fig. 5A and see domains: an N-terminal dinucleotide-binding domain with a
Fig. 9B), so the binding of the sulfate likely results in subunit A classical Rossmann fold (Domain I), a C-terminal catalytic
being in a closed conformation.
domain containing an ␣/␤-fold (Domain II), and an all-helical
Comparison with Known Protein Structures—When we sent domain formed by five ␣-helices (␣10–␣14) and one short turn
our model of the LysDH monomer to the Dali server (34) seek- helix (␥3) (Domain III). Interestingly, the main chain coordi-
ing proteins with similar structures, we found that the coen- nates of the LysDH monomer were closely related to those of
zyme-binding domains (Rossmann fold) of a large number of the architecture formed by Domains I and II in the saccharo-
8450 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of L-Lysine Dehydrogenase
reductase, in which dimer interac-
tions are found between residues in
the loop connecting ␤10 and ␤11
to their 2-fold symmetry-related
counterparts (Fig. 6B). By contrast,
LysDH dimer interactions were
between residues around ␣11, ␣12,
and loop 147–157 in both subunits,
as mentioned above (Fig. 4A). Thus,
the presence or absence of Domain
III substantially affects the arrange-
ment of the subunits in these two
enzymes.
Cofactor Binding—The following
description of cofactor binding in
the active site is based on the struc-
ture of subunit A. The electron den-
sity corresponding to the NAD
coenzyme bound within the active
site was very clear, which enabled us
to place the ligand with reasonable
accuracy. The map enabled clear
positioning of the adenine ring,
identification of the C2Ј-endo-con-
formation of the two ribose rings,
clear definition of the backbone
phosphate groups, and assignment
of an anti-conformation to the gly-
cosidic bond linking the nicotin-
amide ring and its associated ribose
moiety. N-3 and N-6 of the adenine
base form hydrogen bonds with the
side chains of Asp29 and Asp48,
respectively (Fig. 8), and the
hydroxyl groups of the adenine
ribose interact with the side chains
of Asp29 and Asn34. 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 (36). In
LysDH, this motif occupies the 7th–
FIGURE 7. Amino acid sequence alignment of three LysDHs and saccharopine reductase. PH, P. horikoshii;
12th amino acids from the N termi-
nus (Fig. 7). In addition, the nicotin-
amide phosphate interacts with
Ile11, Ser161, and a water molecule
(position 2036). The hydroxyl
GS, G. stearothermophilus; AT, A. tumefaciens; MG SPRD, M. grisea saccharopine reductase. Sequences were
aligned using ClustalW (38). Asterisks indicate conserved residues. The residues involved in binding the sulfate
ion and NAD are shown in blue and red, respectively. The ␣-helices (␣1–␣16; yellow) and ␤-strands (␤1–␤16;
purple) in P. horikoshii LysDH are shown above the alignment.
pine reductase (Fig. 6A). Domain III is absent in LysDH, groups of the nicotinamide ribose interact with the side chain of
although two ␣-helices from that domain, ␣10 and ␣14, are Asp164 and the main chain amides of Gly71 and Phe93. The
conserved in LysDH as ␣11 and ␣12, respectively. The sequence pattern of hydrogen bonding between the enzyme and NAD is
alignment also showed that 68 residues inserted between ␣10 completed by interactions between the carboxamide moiety of
and ␣14 in the M. grisea saccharopine reductase are not present the nicotinamide ring and the main chain amides of Phe118 and
in P. horikoshii LysDH (Fig. 7). A similar deletion of the residues Ala119, the main chain oxygen of cis-Pro120, and the side chain
was observed in the sequences of the G. stearothermophilus and of Thr299. These hydrogen bonds cause the glycosidic bond
A. tumefaciens LysDHs (Fig. 7).
between the nicotinamide ring and its associated ribose moiety
The quaternary structural arrangement of P. horikoshii to be in the anti-conformation. In general, NAD(P)H-depen-
LysDH is totally different from that of M. grisea saccharopine dent dehydrogenases show ether pro-R or pro-S stereospecificity
MARCH 12, 2010•VOLUME 285•NUMBER 11
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Crystal Structure of L-Lysine Dehydrogenase
for hydrogen removal from C-4 of the nicotinamide moiety of of this structure (Protein data bank code 1E5Q) onto the struc-
NAD(P)H. NAD binds to pro-R-specific enzymes in an anti- ture of LysDH enabled us to compare the amino acid residues
conformation and to pro-S-specific enzymes in a syn-confor- involved in substrate binding. On the basis of the orientation of
mation (37), which suggests that P. horikoshii LysDH makes use the saccharopine, we modeled the L-lysine molecule into the
of pro-R-specific hydride transfer (type A), as has been active site of P. horikoshii LysDH (Fig. 9A) and then minimized
described for A. tumefaciens LysDH (7).
the energy of the complex using Swiss-PdbViewer. Within this
Active Site and Insight into the LysDH Reaction—The crystal structure, the carboxyl group of L-lysine forms a salt bridge with
structure of M. grisea saccharopine reductase in complex with the side chain of Arg226 and a hydrogen bond with the main
saccharopine and NADPH has been determined. Superposition chain amide proton of Gly145, and the C-␣ amino group is sit-
uated within hydrogen-bonding distance of the side chain of
Thr224 (Fig. 9B). These three residues are strictly conserved in
saccharopine reductase as Arg247, Thr245, and Gly157, respec-
tively, and hold saccharopine in the active site in a similar man-
ner (Fig. 9A). In saccharopine reductase, moreover, the side
chain of Trp174 is engaged in a hydrophobic interaction with
the carbon skeleton of saccharopine. This residue is also con-
served as Trp160 in LysDH. By contrast, Ser99, Tyr100, and
Arg224 in saccharopine reductase, which interact with the car-
boxyl group of the glutamate moiety of saccharopine, are com-
pletely absent in LysDH, and superposition of the two enzymes
results in a serious steric clash between the glutamate moiety of
saccharopine and the side chain of Phe93 in LysDH (Fig. 9A).
This is consistent with our observation that LysDH does not
utilize saccharopine as a substrate. The most important differ-
ence between the two enzymes is that Asp126 in saccharopine
reductase is replaced by Ala119 in LysDH. In our model, the
main chain oxygen of Ala119 forms a hydrogen bond with the
nitrogen atom of the L-lysine ⑀-amino group (Fig. 9B). As a
FIGURE 8. Stereo representation of NAD bound to P. horikoshii LysDH.
Residues that interact with NAD are labeled. The networks of hydrogen
bonds are shown as dashed lines. The final ␴A-weighted 2Fo Ϫ Fc electron
density map for NAD is shown at the 1␴ level. Atoms are colored as
result, the pro-R-hydrogen at prochiral C-6 of L-lysine is placed
in front of the re-face of the nicotinamide ring, which enables
the pro-R-specific proton transfer to NAD. This observation is
described for Fig. 3.
in good agreement with the previ-
ous finding that A. tumefaciens
LysDH abstracts stereospecifically
the ⑀-pro-R-hydrogen of L-lysine
(6).
In the reverse reaction catalyzed
by saccharopine reductase (forma-
tion of glutamate), Asp126 is thought
to be involved in the hydrolysis of
the Schiff base intermediate (22),
which requires nucleophilic attack
by a water molecule on C-6 of sac-
charopine, and the nucleophilicity
of this water molecule is assumed to
be enhanced by interactions with
the side chain of Asp126. However,
the catalytic residue (Asp126) of
M. grisea saccharopine reductase
has been identified only from the
structural information; it has not yet
been confirmed by site-directed
mutagenesis. We therefore used
site-directed mutagenesis to search
for a catalytic residue in the LysDH
active site. We found Glu168 to be
FIGURE 9. A, comparison of the active-site pocket of P. horikoshii LysDH with that of M. grisea saccharopine
reductase. B, proposedmodelforL-lysinebinding. P. horikoshiiLysDHandM. griseasaccharopinereductaseare
shown in white and green, respectively. L-Lysine (cyan) and saccharopine (yellow) molecules are shown as stick
models. Atoms are colored as described for Fig. 3. WAT, water.
the only residue close enough to C-6
of L-lysine to serve that purpose
8452 JOURNAL OF BIOLOGICAL CHEMISTRY
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Crystal Structure of L-Lysine Dehydrogenase
(Fig. 9A). Aside from Glu168, there are no other obvious resi-
dues in the active site that might facilitate the proton transfer
expected to occur during catalysis. For that reason, we con-
structed E168Q and E168A mutants. Notably, however, the
kinetic analysis of the two mutants indicated that their catalytic
activities were nearly the same as that of the wild-type enzyme,
although the Km values for L-lysine were slightly increased to
2.0–2.3 mM. In addition, Glu168 is conserved as Glu174 in
A. tumefaciens LysDH but is replaced by His181 in G. stearo-
thermophilus LysDH (Fig. 7). Apparently, Glu168 is not essential
for enzyme catalysis. Among the residues located within the
substrate-binding pocket, Tyr156 and Arg226 are strictly con-
served in LysDHs and saccharopine reductase. We therefore
also constructed Y156F and R226K mutants and observed that
8. Misono, H., Hashimoto, H., Uehigashi, H., Nagata, S., and Nagasaki, S.
(1989) J. Biochem. 105, 1002–1008
9. Hashimoto, H., Misono, H., Nagata, S., and Nagasaki, S. (1990) Agric. Biol.
Chem. 54, 291–294
10. Ohshima, T., and Soda, K. (1989) Trends Biotechnol. 7, 210–214
11. Hummel, W., and Kula, M. R. (1989) Eur. J. Biochem. 184, 1–13
12. Satoh, A., Kim, J. K., Miyahara, I., Devreese, B., Vandenberghe, I., Hacisa-
lihoglu, A., Okajima, T., Kuroda, S., Adachi, O., Duine, J. A., Van Beeumen,
J., Tanizawa, K., and Hirotsu, K. (2002) J. Biol. Chem. 277, 2830–2834
13. Durham, D. R., and Perry, J. J. (1978) J. Bacteriol. 134, 837–843
14. Limburg, J., Mure, M., and Klinman, J. P. (2005) Arch. Biochem. Biophys.
436, 8–22
15. Nozaki, M. (1987) Methods Enzymol. 142, 650–655
16. Murray, M. G., and Thompson, W. F. (1980) Nucleic Acids Res. 8,
4321–4325
17. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254
these mutations completely abolished the activity of the 18. Kusakabe, H., Kodama, K., Kuninaka, A., Yoshino, H., Misono, H., and
enzyme. In our model, the side chain hydroxyl group of Tyr156
Soda, K. (1980) J. Biol. Chem. 255, 976–981
and the guanidium group of Arg226 (NH2) are estimated to be
19. Ohshima, T., Misono, H., and Soda, K. (1978) J. Biol. Chem. 253,
5719–5725
7.8 Å from C-6 of L-lysine, whereas the corresponding groups in
20. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326
Tyr170 and Arg247 (NH1) of saccharopine reductase are 7.1–7.5
21. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D 55,
Å from C-6 of saccharopine. These distances preclude the
involvement of these two residues in the hydride transfer.
Instead, it appears that Tyr156 and Arg226 play essential roles in
the binding of substrates to LysDHs.
849–861
22. Terwilliger, T. C. (2000) Acta Crystallogr. Sect. D 56, 965–972
23. McRee, D. E. (1999) J. Struct. Biol. 125, 156–165
24. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr.
Sect. D 53, 240–255
Although the mechanism underlying the catalytic reaction of
P. horikoshii LysDH remains unclear, this study provides the
first structural insight into substrate stereo recognition and
hydride transfer by LysDH. The structure of the P. horikoshii
LysDHꢀNADꢀsubstrate analog ternary complex should be a use-
ful focus for further investigation.
25. Bru¨nger, 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 54, 905–921
26. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M.
(1993) J. Appl. Crystallogr. 26, 283–291
27. Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K., and Noble, M.
(2002) Acta Crystallogr. Sect. D 58, 1955–1957
Acknowledgments—We thank Drs. N. Igarashi, N. Matsugaki, and S.
Wakatsuki for help in data collection at Photon Factory beamline
BL5A. We also thank Drs. S. Kawamura, T. Araki, and T. Torikata for
extremely helpful support.
28. Potterton, L., McNicholas, S., Krissinel, E., Gruber, J., Cowtan, K., Emsley,
P., Murshudov, G. N., Cohen, S., Perrakis, A., and Noble, M. (2004) Acta
Crystallogr. Sect. D 60, 2288–2294
29. Matthews, B. W. (1968) J. Mol. Biol. 33, 491–497
30. Hashimoto, H., Misono, H., Nagata, S., and Nagasaki, S. (1989) J. Biochem.
106, 76–80
REFERENCES
31. Qi, G., Lee, R., and Hayward, S. (2005) Bioinformatics 21, 2832–2838
32. Lee, R. A., Razaz, M., and Hayward, S. (2003) Bioinformatics 19,
1290–1291
1. Misono, H., and Nagasaki, S. (1982) J. Bacteriol. 150, 398–401
2. Heydari, M., Ohshima, T., Nunoura-Kominato, N., and Sakuraba, H.
(2004) Appl. Environ. Microbiol. 70, 937–942
33. Hayward, S., and Berendsen, H. J. (1998) Proteins 30, 144–154
34. Holm, L., and Sander, C. (1998) Nucleic Acids Res. 26, 316–319
35. Johansson, E., Steffens, J. J., Lindqvist, Y., and Schneider, G. (2000) Struc-
ture 8, 1037–1047
3. Ruldeekulthamrong, P., Maeda, S., Kato, S., Shinji, N., Sittipraneed, S.,
Packdibamrung, K., and Misono, H. (2008) BMB Rep. 41, 790–795
4. Ohshima, T., and Soda, K. (2000) in Stereoselective Biocatalysis (Patel,
R. N., ed) pp. 877–902, Marcel Dekker, Inc., New York
36. Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., and Stillman, T. J. (1992) J.
Mol. Biol. 228, 662–671
5. Bu¨rgi, W., Richterich, R., and Colombo, J. P. (1966) Nature 211, 854–855
6. Misono, H., Yoshimura, T., Nagasaki, S., and Soda, K. (1990) J. Biochem.
107, 169–172
37. Benner, S. A. (1982) Experientia 38, 633–636
38. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res.
22, 4673–4680
7. Hashimoto, H., Misono, H., Nagata, S., and Nagasaki, S. (1989) Agric. Biol.
Chem. 53, 1175–1176
MARCH 12, 2010•VOLUME 285•NUMBER 11
JOURNAL OF BIOLOGICAL CHEMISTRY 8453

Protein Structure and Folding:
First Crystal Structure of l-Lysine
6-Dehydrogenase as an NAD-dependent
Amine Dehydrogenase
Kazunari Yoneda, Junya Fukuda, Haruhiko
Sakuraba and Toshihisa Ohshima
J. Biol. Chem. 2010, 285:8444-8453.
doi: 10.1074/jbc.M109.084384 originally published online January 7, 2010
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