A second novel dye-linked L-proline dehydrogenase
complex is present in the hyperthermophilic archaeon
Pyrococcus horikoshii OT-3
Ryushi Kawakami
1
, Haruhiko Sakuraba
1
, Hideaki Tsuge
2,3
, Shuichiro Goda
1
, Nobuhiko Katunuma
2
and Toshihisa Ohshima
1
1 Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan
2 Institute for Health Science, Tokushima Bunri University, Japan
3 The Institutes for Enzyme Research, University of Tokushima, Japan
Dye-linked dehydrogenases (dye-DHs) catalyze the oxi-
dation of various amino acids, organic acids, amines
and alcohols in the presence of artificial electron accep-
tors such as 2, 6-dichloroindophenol (DCIP) and ferri-
cyanide. Although dye-DHs show a high potential for
use as specific elements in biosensors [1], their low
stability has thus far precluded their use in practical
applications and limited our ability to obtain detailed
information about their structures and functions.
Recently, however, much attention has been paid to
the isolation and characterization of enzymes from
hyperthermophilic archaea, as these organisms repre-
sent a source of extremely stable enzymes. Indeed, we

structure,
while PDH2 has an abcd structure. PDH2 was found to be similar to the
dye-linked l-proline dehydrogenase complex from Thermococcus profundus,
but PDH1 is a different type of enzyme. After production of the enzyme
in Escherichia coli, high-performance liquid chromatography showed the
PDH1 complex to contain the flavins FMN and FAD as well as ATP.
Gene expression and biochemical analyses of each subunit revealed that
the b subunit bound FAD and exhibited proline dehydrogenase activity,
while the a subunit bound ATP, but unlike the corresponding subunit in
the T. profundus enzyme, it exhibited neither proline dehydrogenase nor
NADH dehydrogenase activity. FMN was not bound to either subunit,
suggesting it is situated at the interface between the a and b subunits.
A comparison of the amino-acid sequences showed that the ADP-binding
motif in the a subunit of PDH1 clearly differs from that in the a subunit
of PDH2. It thus appears that a second novel dye-linked l-proline dehy-
drogenase complex is produced in P. horikoshii.
Abbreviations
dye-DH, dye-linked dehydrogenase; DCIP, 2,6-dichloroindophenol; dye-
L-proDH, dye-linked L-proline dehydrogenase; dye-NADHDH, dye-
linked NADH dehydrogenase.
4044 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS
(dye-l-proDH) [3,4], and found these enzymes to be
highly stable and to exhibit a high potential for appli-
cation in amino-acid analyses.
Dye-l-proDH catalyzes the oxidation of l-proline to
D
1
-pyrroline-5-carboxylate in the presence of DCIP.
We first identified this enzyme in the hyperthermo-
philic archaeon Thermococcus profundus DSM9503 [4].

activity staining as described in the ‘Experimental
procedures.’ We observed two separate activity bands
with P. horikoshii and T. peptonophilus; one band
with T. profundus, P. furiosus and P. abyssi; and no
activity bands with T. litoralis (data not shown).
These results suggest that P. horikoshii and T. peptono-
philus each produce two distinguishable forms of dye-
l-proDH. Because its genome has been sequenced
completely [5], we chose to purify the enzymes from
P. horikoshii.
Purification of PDH1 and PDH2 from P. horikoshii
OT-3 and identification of the encoding genes
The steps used to isolate PDH1 and PDH2 from
P. horikoshii OT-3 are summarized in Tables 1 and 2,
respectively. We succeeded in separating the two
enzymes using Butyl Toyopearl column chromatogra-
phy (Fig. 1), and were able to further purify them
using the additional steps listed. Gel filtration
Table 1. Purification of PDH1 from P. horikoshii.
Total protein (mg) Total activity (U) Specific activity (UÆmg
)1
) Yield (%) Fold
Crude extract 4550 65.1 0.0143 100 1.0
Ammonium sulfate
fractionation
3600 58.6 0.0163 90 1.1
DEAE Toyopearl 1040 51.7 0.0497 79 3.5
Butyl Toyopearl 295 7.40 0.0251 11 1.8
Cellulofine HAp 13.3 5.90 0.444 9 31.0
UnoQ 2.63 2.47 0.939 4 65.7

) structure. The N-terminal amino-acid sequence
of the a1 subunit was determined to be
MRPLDLTEKR, which corresponds to the underlined
amino-acid sequence in ML
MRPLDLTEKR from the
putative protein encoded by the predicted open reading
frame (ORF; PH1363) based on the genome analysis.
This means that ATG, which was situated 7 bp down-
stream from the 5¢-terminus of the predicted ORF, is the
proper initial codon for the a1 gene. The N-terminal
amino-acid sequence of the b1 subunit was determined
to be MLPEKSEIVV, which corresponds to that of the
predicted PH1364 gene product. The a1 and b1 genes
were arranged in tandem (a1 –b1) and were estimated to
encode proteins with molecular masses of 55 316 and
42 685 Da, respectively.
On the other hand, SDS ⁄ PAGE analysis of purified
PDH2 showed four bands (data not shown). The
molecular masses of the a, b, c and d subunits of
PDH2 (designated a2, b2, c2 and d2) were about 52,
46, 20 and 8 kDa, respectively. Although the four dif-
ferent subunits together have a mass of 126 kDa, only
101 kDa has been determined by gel filtration. On the
other hand, SDS ⁄ PAGE after Superose 6 chromato-
graphy showed that all four subunits were present
proportionally in the active fractions. The molecular
ratio of the subunits of PDH2 was determined to be
1 : 1 : 1 : 1 using the same method used for PDH1.
This suggests that the enzyme has a heterotetrameric
(abcd) structure. The N-terminal amino-acid

ively [3]. Identification of the genes encoding the four
subunits of PDH2 clearly demonstrates that PDH2 is
similar to the T. profundus dye-l-proDH complex.
In the present study, the genes encoding the PDH1
subunits were found to form an operon (a1–b1), and
similar gene clusters have been observed in the
genomes of P. furiosus (PF1245–PF1246) and P. abyssi
(PAB1842–PAB1843). A gene cluster like that formed
by the PDH2 genes (a2–c2–d2–b2) is also distributed
in these organisms [3]. Together with the results of
activity screening, these observations suggest that both
the a
4
b
4
and abcd dye-l-proDHs are widely distri-
buted within the order Thermococcales in the archaeal
Fig. 1. Elution profile obtained with Butyl Toyopearl chromatogra-
phy. Enzyme solution was applied on a Butyl Toyopearl column and
eluted with a linear gradient of 40–0% ammonium sulfate in buffer
A. The squares and circles show the activity and A
280
, respectively.
FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al.
4046 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS
domain, though their expression patterns differ from
one another depending up the hyperthermophilic
species examined and cultivation conditions used.
Expression of the PDH1 gene and purification
of the recombinant enzyme

under various pH conditions was examined while incu-
bating the enzyme at 50 °C for 30 min. The enzyme
lost no activity between pH 5.0 and 10.0 (Fig. 2C),
and the optimum pH was determined to be 7.5
(Fig. 2D).
Table 3. Purification of recombinant PDH1 from E. coli.
Total protein (mg) Total activity (U) Specific activity (UÆmg
)1
) Yield (%) Fold
Crude extract 2280 111 0.0487 100 1.0
Heat treatment 400 279 0.698 251 14.3
Butyl Toyopearl 62 113 1.82 102 37.4
Superdex 200 50 105 2.10 95 43.1
Fig. 2. (A) Thermostability of PDH1. The
enzyme was incubated at 90 °C, and the
residual activity was measured at the indica-
ted times. (B) Effect of temperature on
PDH1 activity. The enzyme activity was
measured at various temperatures between
50 and 95 °C. (C) pH stability of PDH1. The
enzyme was incubated at 50 °C for 30 min
in buffers of various pH, after which residual
activity was measured. (D) Effect of pH on
PDH1 activity. The enzyme activity was
measured at various pHs ranging from 6
and 9. The buffers used were Mes ⁄ NaOH
(pH 6.0–7.0), Hepes ⁄ NaOH (pH 7.0–7.5) and
Tris ⁄ HCl (pH 7.5–9.0).
R. Kawakami et al. FAD, FMN and ATP-containing amino-acid dehydrogenase
FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS 4047

unit of PDH1 contained an ADP-binding motif [6],
which was well conserved in the b subunit of the abcd-
type enzymes (Fig. 4). In addition, expression of the b1
gene in E. coli using a pET15b ⁄ b1 system revealed that,
like the b subunit of the abcd-type enzyme [3], the b1
subunit is capable of catalyzing l-proline dehydrogena-
tion by itself (data not shown). This suggests that the b1
subunit of PDH1 has the same function as that des-
cribed for the b subunit of T. profundus dye-l-proDH;
that is, it catalyzes the first reaction of the incorporation
of electrons from l-proline into the electron transfer sys-
tem [3]. We previously reported that the T. profundus
enzyme exhibits dye-NADHDH activity as well as
l-proline dehydrogenase activity, and functional analy-
sis of each subunit showed that it is the a subunit that
catalyzes the dye-NADHDH reaction [3]. We attempted
to detect dye-NADHDH activity using the PDH1 com-
plex, but found none. In addition, when we expressed
Fig. 3. Amino-acid sequence alignment of
the a subunits of PDH1, PDH2 and T. pro-
fundus dye-
L-proDH: alpha1, a1 subunit of
PDH1; alpha2, a2 subunit of PDH2; and
pdhA, a subunit of T. profundus dye-
L-
proDH. Asterisks(*) represent conserved
residues. ADP-binding motifs [6] are boxed.
FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al.
4048 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS
the a1 gene in E. coli using a pET11a ⁄ a1 system, the

and b1 subunits separately produced in E. coli cannot
bind FMN.
While carrying out the procedure to identify the fla-
vin compounds, we observed an unexpected signal that
had about a 4-min retention time on the TSKgel ODS-
80Tm column (Fig. 5A). This signal corresponds to
that of the ATP standard, and when a sample was
injected together with authentic ATP, an enhancement
of the peak was observed. The presence of ATP was
also demonstrated using an Asahipak GS-320HQ col-
umn (data not shown). The ATP content was about
4 mol of ATP per mol of enzyme complex, and
ATP was present in the native enzyme as well as in the
separately produced a1 subunit (Fig. 5C,D), suggesting
that the enzyme complex contains 1 mol of ATP per
mol of a1 subunit. The T. profundus abcd-type enzyme
contains 2 mol of FAD per mol of enzyme complex
[3]. As mentioned above, the a subunit of the enzyme
has two ADP-binding motifs, and in a previous report
we showed it to also contain 1 mol of FAD per mol of
a subunit [3]. In this subunit, any other prosthetic
groups than FAD were not detected [3]. As the a sub-
unit exhibited dye-NADHDH activity, we supposed
that one ADP-binding motif mediates FAD binding
and the other is responsible for the dye-NADHDH
Fig. 4. Amino-acid sequence alignment of
the b subunits of PDH1, PDH2 and T. pro-
fundus dye-
L-proDH: beta1, b1 subunit of
PDH1; beta2, b2 subunit of PDH2; and

trophic bacterium W3A1 reportedly produces an
ADP-containing oxidoreductase, trimethylamine dehy-
drogenase, but not an ATP-containing dehydrogenase,
and the function of ADP in trimethylamine dehydro-
genase remains unknown [16]. Similarly, the catalytic
properties of PDH1 can be interpreted without consid-
ering the function of ATP. It may be that this ATP
has a stabilizing effect on the protein, or that it plays
an unknown regulatory role, as has been suggested for
ADP in trimethylamine dehydrogenase. To the best of
our knowledge, this is the first example of an oxido-
reductase complex that contains an ATP.
In general, dye-DHs play important roles in the
incorporation of electrons from a substrate into
the electron-transfer system. In the present study, the
PDH1 complex was found to contain FAD, FMN and
ATP. That PDH1 is totally different from any known
electron-transfer flavoprotein that utilizes both FAD
and FMN as cofactors, suggests this enzyme may
employ a novel electron transfer pathway from l-pro-
line to the electron-transfer system. Our goal is to bet-
ter understand the relationship between the structure
and function of each subunit of this enzyme and the
physiological functions of the unique prosthetic
groups. An X-ray reflection analysis of PDH1 is now
in progress. These are essential steps in an effort to
achieve the practical application of dye-l-proDHs.
Experimental procedures
Materials
DCIP and l-proline were purchased from Nacalai Tesque

4
Æ7H
2
O, 0.07 g of CaCl
2
Æ2H
2
O, 0.02 g of FeCl
3
Æ6H
2
O,
Fig. 5. HPLC analyses of the prosthetic groups. Elution profiles of
extracts of recombinant PDH1 (A), the b subunit of PDH1 (B), the a
subunit of PDH1(C), native PDH1 prepared from P. horikoshii cells
(D), and the standard mixture (E).
FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al.
4050 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS
1.8 mg of MnCl
2
Æ4H
2
O, 4.5 mg of Na
2
B
4
O
7
Æ10H
2

)30 °C.
Enzyme assay and protein determination
Enzyme activity was spectrophotometrically assayed as
described previously using a Shimadzu UV-1200 spectro-
photometer equipped with a thermostat [3]. Protein
concentrations were determined using Bradford method;
bovine serum albumin served as the standard [17].
Purification of dye-L-proDHs from P. horikoshii
OT-3
P. horikoshii cells (wet weight, about 40 g) obtained from
50 L of medium were suspended in 360 mL of buffer A
and disrupted by ultrasonication. After centrifugation
(20 000 g, 20 min) to remove any remaining intact cells and
the cell debris, the supernatant was used as the crude
extract. Ammonium sulfate was added to the extract to
40% saturation, after which it was stirred at 4 °C for 1 h
and centrifuged again (20 000 g, 20 min), and additional
ammonium sulfate was added to the resultant supernatant
containing the enzyme to bring it up to 70% saturation.
Then after 1 h the solution was centrifuged, and the pre-
cipitant, which contained the enzyme, was suspended in
buffer A and dialyzed against the same buffer.
The enzyme-containing solution was then loaded onto a
DEAE Toyopearl column (4.8 · 11 cm; Tosoh, Tokyo,
Japan) equilibrated with buffer A. After washing the col-
umn with the same buffer, the enzyme was eluted with a
1.3-L linear gradient of 0 to 0.2 m NaCl in buffer A. The
active fractions were collected, and the sample was brought
up to 40% saturation with ammonium sulfate and then
loaded onto a Butyl Toyopearl column (3.6 · 10 cm;

and cloning of the enzyme genes
To obtain the genomic DNA containing the PDH1 and
PDH2 genes, P. horikoshii cells were first cultured as des-
cribed above, filtered to remove the sulfur powder and then
centrifuged (10 000 g, 15 min). The cells were then washed
twice with 3% NaCl, and the OT-3 genomic DNA was pre-
pared by the method of Murray and Thompson [18]. To
avoid any nucleotide incorporation errors we did not use
the PCR method to clone the PDH1 and PDH2 genes.
Instead, two oligonucleotide probes (5¢-ATGAGACC
TCTAGATCTAAC-3¢ for the PDH1 gene and 5¢-TATA
TTTAGGTGGAAATTGT-3¢ for the PDH2 gene) were
synthesized based on the DNA sequence in the P. horikoshii
genome database, after which 1.5-pmol samples were labe-
led with [ c -
32
P]ATP (1.85 MBq) using T4 polynucleotide
kinase (10 U), purified on a ProbeQuant
TM
G-50 micro-
column, and used as specific probes for southern and
colony hybridizations.
For preparation of the PDH1 gene, the genomic DNA
was digested with SphI and KpnI; for the PDH2 gene it
was digested with BamHI and SphI, and the resultant
fragments were separated on 0.8% agarose gels. Approxi-
mately 8.0 kbp of fragments digested with SphI and KpnI
and 7.5 kbp with BamHI and SphI were extracted from
the gels and inserted between the SphI and KpnI sites of
plasmid pUC19 and the BamHI and SphI sites of pUC18,

for the PDH1 gene: 5¢-AGGGATGCATATGAGACCT
CTAGATCTAAC-3¢ and 5¢-AGGCCCCGGGTCACCTC
CTAGCTAGAATTC-3¢ for a1; and 5¢-AGGTGATC
ATATGCTTCTAGAGAAGAGTGAAATA-3¢ and 5¢-AG
AGGATCCTCAGCCCATTTGGAGGGCGG-3¢ for b1.
In each case, the forward primer introduced a unique NdeI
restriction site that overlapped the 5¢-initiation codon, and
the reverse primer introduced a unique SmaIorBamHI
restriction site proximal to the 3¢-end of the termination
codon. PCR was carried out using pPDH1 as the template,
after which the amplified fragments were digested with NdeI
and SmaI for a1 and with NdeI and BamHI for b1. For
ligation to a1, the plasmid pET11a was digested with
BamHI, blunted and then further digested with NdeI. For
ligation to b1, the plasmid pET11a was digested with NdeI
and BamHI. The a1 and b 1 gene fragments were introduced
into pET11a after linearizing it with NdeI and blunted-
BamHI to generate pET11a ⁄ a1 and with NdeI and BamHI
to generate pET11a ⁄ b1, respectively. pET11a ⁄ a1 was then
digested with ClaI, blunted and further digested with SphI.
The resultant fragment containing a1 and the T7 promoter
was introduced into pET11a ⁄ b1 digested with SphI and
BglII (the BglII site had already been blunted) to generate
the expression plasmid pEPDH1, which was then used to
transform E. coli strain BL21 CodonPlus RIL (DE3) cells.
The transformants were grown for 8 h at 37 ° C in SB med-
ium (1.2% tryptone peptone, 2.4% yeast extract, 1.25%
K
2
HPO

. The active fractions were pooled and dia-
lyzed against buffer B. All buffers used in the purification
were degassed before use.
Subunit gene expression and product purification
Separate expression systems for the a1 and b1 subunits
were constructed to determine the function of each subunit.
For production of the a1 subunit, E. coli strain BL21
CodonPlus RIL (DE3) cells transformed with pET11a ⁄ a1
were grown for 6 h at 37 °C in SB medium in the presence
of ampicillin (100 lgÆml
)1
), after which IPTG was added to
1mm, and cultivation was continued for an additional 3 h.
The cells were then collected by centrifugation (10 000 g,
20 min), suspended in 10 mm potassium phosphate, pH 7.0
containing 1 mm DTT, and disrupted by sonication. After
centrifugation (20 000 g, 20 min), the supernatant was col-
lected and heated at 80 °C for 10 min and centrifuged
(20 000 g, 20 min) again to remove the denatured proteins.
Ammonium sulfate was added to the supernatant to 20%
saturation, after which the enzyme solution was applied to
a Butyl Toyopearl column (2.6 · 6 cm) equilibrated with
buffer B supplemented with 20% ammonium sulfate. After
washing the column with the same buffer, the enzyme was
eluted with a 300-mL linear gradient of 20–0% ammonium
sulfate in buffer B. The active fractions were pooled and
used as the purified enzyme preparation.
When a pET11a ⁄ b1 expression system was used for the
production of the b1 subunit, the enzyme produced was
found mainly in the insoluble fraction as an inclusion body.

groups
Flavin compounds and other prosthetic groups from the
enzyme were extracted with 1% (w ⁄ v) perchloric acid. The
solution was then centrifuged, and after neutralizing the
supernatant with K
2
CO
3
, it was subjected to HPLC using
TSKgel ODS-80Tm (Tosoh) and Asahipak GS-320HQ
(Shodex, Tokyo) columns. An isocratic elution (10 min)
with 10 mm potassium phosphate, pH 6.0 followed by a lin-
ear gradient (30 min) between 0 and 70% methanol in the
same solution was used for the TSKgel ODS-80Tm column.
An isocratic elution with 200 mm sodium phosphate,
pH 5.0 was used for the Asahipak GS-320HQ column. The
flow rate was 1.0 mLÆmin
)1
, and the absorbance at 260 nm
of the effluent from the column was monitored.
Polyacrylamide gel electrophoresis and molecular
mass determination
Native PAGE was carried out with 7.5% polyacrylamide
gel according to the method of Davis [20]. Activity staining
was carried out at 50 °C, as previously described [4].
SDS ⁄ PAGE was carried out using 15% polyacrylamide gel
containing 0.1% SDS according to the method of Leammli
[21]. The subunit molecular mass was determined using
eight marker proteins (New England Biolabs).
The molecular masses of the native enzymes were deter-

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FAD, FMN and ATP-containing amino-acid dehydrogenase R. Kawakami et al.
4054 FEBS Journal 272 (2005) 4044–4054 ª 2005 FEBS