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J. Biol. Chem., Vol. 277, Issue 26, 23821-23827, June 28, 2002
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From the Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan
Received for publication, March 21, 2002, and in revised form, April 16, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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A protein containing a heme-binding PAS
(PAS is from the protein names in which imperfect repeat
sequences were first recognized: PER, ARNT, and
SIM) domain from Escherichia coli has been
implied a direct oxygen sensor (Ec DOS) enzyme. In the
present study, we isolated cDNA for the Ec DOS
full-length protein, expressed it in E. coli, and examined
its structure-function relationships for the first time. Ec
DOS was found to be tetrameric and was obtained as a 6-coordinate low
spin ferric heme complex. Its Heme proteins and enzymes perform a broad range of functions. Well
known examples include O2 storage with myoglobin,
O2 carriage with hemoglobin, mediators of electron transfer
with cytochromes, and catalytic activation of heme ligands with P450s
and peroxidases (1-3). Recently, a new class of heme enzymes involved
in intramolecular signal transduction is emerging, known as heme-based
sensors (4-6). Almost of all the heme-based sensors contain two
different functional domains as follows: one is an N-terminal heme
domain, which acts a sensor, and the other is a catalytic domain such
as a histidine kinase or a soluble guanylate cyclase. These heme sensor
enzymes use the heme for mediating transcriptional and regulatory
events associated with the presence of gaseous molecules such as CO, NO, and O2 (4-6). In these enzymes, the ligand association
or dissociation from the heme iron leads to protein conformational changes, which transmit signals to the other domain where they initiate
catalytic function or DNA binding. For example, the
CooA1 protein from
Rhodospirillum rubrum is a CO sensor heme protein that
regulates the expression of the coo genes associated with CO-dependent growth (Refs. 7 and 8 and references therein). Soluble guanylate cyclase is an NO sensor heme protein that regulates conversion of 5'-GTP to the intracellular second messenger, cGMP (Refs.
9 and 10 and references therein). Hem-AT-Bs and Hem-AT-Hs are oxygen
sensors in which the hemes are thought to mediate signal transduction
for methylation of the chemotaxis proteins (11, 12).
The Fix proteins, FixL and FixJ, of Rhizobium
meliloti are well characterized as biological oxygen sensors and
regulate the expression of the nitrogen fixation genes of a plant
symbiotic bacterium, Sinorhizobium meliloti (Refs. 13 and 14
and references therein). Dissociation of O2 from FixL
Fe(II) heme initiates its histidine kinase function in another subunit
in FixL. The phosphate group transfers to an Asp residue of FixJ.
Phosphorylated FixJ then acts as a transcriptional activator of the
nifA and fixK genes, which controls the
expression of nitrogen fixation genes and a high affinity terminal
oxidase complex, respectively. The N-terminal portion of FixL consists
of a heme sensory domain whose sequence and tertiary structure
characterize it as a PAS domain. PAS is an acronym formed from the
names of the proteins in which imperfect repeat sequences were first
recognized as follows: the Drosophila period clock protein
(PER) (15), vertebrate aryl hydrocarbon receptor nuclear
translocator (ARNT) (16), and Drosophila single-minded protein (SIM) (17).
Based on the sequence homology of the heme sensor enzyme, FixL,
Gilles-Gonzales group identified a heme domain with a PAS sequence from
Escherichia coli (designated Ec DOS for E. coli direct oxygen sensor) (18). They characterized the
physicochemical properties of the isolated heme domain of
Ec DOS. However, they did not report cloning of the
full-length protein or characterize its catalytic function. The same
groups cloned a phosphodiesterase (PDE) A1 protein from
Acetobacter xylinum (designated AxPDEA1) and found that it
is a key regulator of bacterial cellulose synthesis (19). AxPDEA1
linearizes cyclic bis(3',5')diguanylic acid, an allosteric activator of
the bacterial cellulose synthase. The N-terminal 140 amino acids of
AxPDEA1 contain the heme-binding PAS motif. The PDE activity of the
C-terminal domain of AxPDEA1 has been well characterized (19).
Importantly, AxPDEA1 is highly homologous over its entire length to the
Ec DOS protein (18). Therefore, it was speculated that
Ec DOS would be functionally similar.
In the present study, we report for the first time the isolation of
cDNA for full-length wild type Ec DOS, expression in
E. coli, and characterization of its functions under various
conditions. Mutations at the heme-binding site enabled one of the axial
ligands to the heme to be identified. Importantly, it was found that
Ec DOS has PDE activity toward cAMP, only when the heme is
in the ferrous form. Its activity was strongly inhibited by heme
ligands such as CO and NO and a selective PDE inhibitor. Thus, this
work proves that this enzyme is a PDE with a redox-sensitive heme sensor.
Materials--
The genomic DNA of E. coli JM 109 strain was purchased from Takara Shuzo Co. (Kyoto, Japan).
Oligonucleotides were synthesized by Nihon Gene Research Laboratory
(Tokyo, Japan), GENSET OLIGOS (Sendai, Japan), and ESPEC OLIGO Service
Corp. (Tokyo, Japan). Cloning vector, pBluescript SK II(+), pKF19, and
expression vector, pET28a(+), were purchased from Toyobo (Osaka,
Japan), Takara Shuzo Co., and Novagen (Darmstadt, Germany),
respectively. E. coli competent cells, JM109, XL1-blue (for
cloning), and BL21 (for expression of the protein) were purchased from
Takara Shuzo Co., Novagen, and Stratagene (La Jolla, CA), respectively.
Taq DNA polymerase, dNTP mixture, and other compounds
necessary for PCR were purchased as a Takara Ex TagTM kit
from Takara Shuzo Co. Restriction enzymes and modifying enzymes for DNA
recombination were purchased from Takara Shuzo Co., Toyobo, New England
Biolabs (Beverly, MA), and Nippon Roche K.K. (Tokyo, Japan).
Fluorescence substrate, ant-cAMP, ant-cGMP, and some PDE-specific inhibitors were purchased from Calbiochem.
Bis(p-nitrophenyl) phosphate and p-nitrophenyl
phosphate were purchased from Sigma. Calf intestine alkaline
phosphatase was purchased form Takara Syuzo Co. DEAE-Sephadex was
purchased from Amersham Biosciences. Other chemicals were purchased
from Wako Pure Chemicals (Osaka, Japan).
Cloning of Ec DOS and Construction of the Expression
Plasmids--
The sequences corresponding to Ec DOS and the
Ec DOS-PAS domain were amplified by PCR. E. coli
genomic DNA isolated from E. coli JM109 strain was used as a
template. A sense primer DOS-1 (5' GGA ATT CCA TAT GCG CCA GGA TGC AGA
GGT 3') which introduces an NdeI site and an antisense
primer DOS-2 (5' FCG AGC TCT CAA ATC AAT TGT CGG GTC TGT 3') which
introduces a SacI site were used for DOS-PAS (amino acids
1-133). Sense primer DOS-1 and an antisense primer DOS-3 (5' TCG AGC
TCT CAG ATT TTC AGC GGT A 3') which introduce a SacI site
were used for full-length DOS (amino acids 1-807). The PCR
products were digested by EcoRI and SacI and
inserted in the same sites of the cloning vector, pBluescript SK II(+).
The obtained clones were confirmed by determination of the nucleotide
sequence by Sanger's method using an automatic sequencer DSQ-2000L
(Shimadzu Co., Kyoto, Japan). pBluescript-Ec DOS and
pBluescript-Ec DOS-PAS were digested by NdeI and
SacI and subcloned into E. coli expression
vector, pET 28a(+), which introduces His6 tag at the N
terminus of expressed proteins.
Construction of Expression Plasmids of Ec DOS-PAS
Mutants--
The Ec DOS gene has three unique restriction
enzyme sites near the expected heme-binding site. His-77 is located
between SacII and XmnI, and His-83 is located
between XmnI and PstI. On the other hand, the
pET28a(+) vector has two XmnI sites. Therefore, at first,
the Ec DOS gene was transferred into the pKF19 vector, which
does not have these restriction enzyme sites. Oligonucleotides between
these sites, which have mutation in the position of expected heme-bound
histidine, were synthesized and inserted into the Ec DOS
gene. The sequence was checked by Sanger's method using an automatic
sequencer DSQ-2000L (Shimadzu Co.). Finally, Ec DOS-PAS gene
mutants were transferred into the pET28 vector and expressed by the
same method of Ec DOS full-length wild type.
Expression and Purification of Ec DOS and the Isolated Ec DOS-PAS
Domain--
Ec DOS and the isolated Ec DOS-PAS
domain were expressed in E. coli BL21 cells. Another
plasmid, pGroESL, was co-transformed for expression of chaperon
proteins GroES and GroEL. A single colony was picked and shaken in LB
medium at 37 °C overnight. Then 0.5 ml of overnight culture was put
into 500 ml of TB medium and shaken at 37 °C. The E. coli
cells that contain two kinds of plasmids were cultured in TB medium
containing 50 µg/ml kanamycin and 35 µg/ml chloramphenicol until an
A600 nm up to 1.2 at 37 °C. Then 0.05 mM isopropyl-
The purifications of Ec DOS and the isolated PAS domain were
checked and found to be more than 95% homogeneous by SDS-PAGE. Yields of Ec DOS and the isolated PAS domain were 210 and
610 nmol, respectively, from 1 liter of E. coli culture in
terms of the heme absorbance at 417 nm (18).
Enzymatic Assay--
Ec DOS was incubated at 37 °C
with ant-cAMP or ant-cGMP in a reaction mixture of 500 µl containing
50 mM Tris-HCl buffer (pH 8.5) and 2 mM
MgCl2. To terminate the reaction, Ec DOS was
removed by using an Ultrafree-MC centrifugal filter (Millipore Co.,
Bedford, MA), and 2 units of alkaline phosphatase (Takara Syuzo Co.)
was added and incubated for 1 h at 37 °C. The reaction mixture
was applied to a column of DEAE-Sephadex and washed with water.
Finally, the fluorescence intensity (excitation at 330-350 nm,
emission at 410 nm) of the eluted fraction was measured to determine
the amount of product (20, 21). At least four experiments were conducted to obtain each value; experimental errors are less than 20%.
Assays for bis(p-nitrophenyl) phosphate, and
p-nitrophenyl phosphate were carried out in a solution
containing 50 mM Tris-HCl (pH 8.5), 2 mM
MgCl2, 5 mM substrate, and 0.05-5
µM Ec DOS. The total volume was 2 ml, and this
reaction mixture was incubated at 37 °C for 5 h. Then 1 ml of
0.5 N NaOH was added to the solution to stop the reaction,
and the absorbance at 400 nm was measured.
The reaction of the Ec DOS Fe(III) complex was performed
under aerobic conditions. In the case of the Fe(II) complex, the enzyme
was reduced by sodium dithionite in a glove box under a nitrogen
atmosphere with an O2 concentration of less than 50 ppm (22). Sodium dithionite was removed using a gel filtration column (Sephadex G-25). The enzyme reaction was also carried out in the glove box.
The CO concentration was controlled by addition of CO-saturated buffer
(about 1 mM CO) to the reaction mixture. The NO
concentration was controlled by addition of
(±)-(E)-Methyl-2-[(E)-hydroxyiminol]-5-nitro-6-methoxy-3-hexenamide (Dojindo, Kumamoto, Japan), which releases NO in aqueous solution (23).
(±)-(E)-Methyl-2-[(E)-hydroxyiminol]-5-nitro-6-methoxy-3-hexenamide was dissolved in an adequate amount of Me2SO, and the
Me2SO solution was added to the reaction mixture before
initiating the reaction. The reaction tubes were sealed to prevent the
diffusion of these molecules. All other conditions were the same as
described above.
PDE inhibitors, etazolate, hydrochloride, or
3-isobutyl-1-methylxanthine were dissolved in an adequate amount of
water or ethanol, respectively, and then the solution was added to the reaction mixture to make a final concentration of 2 µM.
All other conditions were the same as described above.
Optical Absorption, Fluorescence, Redox Potential, and CD
Spectra--
Spectral experiments under aerobic conditions were
carried out on Shimadzu UV-1650, UV-2500, and Hitachi U-2010
spectrophotometers maintained at 25 °C by a temperature controller.
Anaerobic spectral experiments were conducted on a Shimadzu UV-160A
spectrophotometer in a glove box. When the heme was reduced by sodium
dithionite, excess dithionite was always removed using Sephadex G-25
column in the glove box. Redox potentials were obtained on the same
spectrometer in the glove box. 2,3,5,6-Tetramethylphenylenediamine,
N-ethylphenazonium ethosulfate, and
2-hydroxy-1,4-naphthaquinone were added as mediators to the sample
before titration. Fluorescence spectra were obtained with Shimadzu
RF-500 and RF-5300PC spectrofluorophotometers. CD spectra were obtained
with a JASCO 2000 CD spectrometer. To ensure that the temperature of
the solution was appropriate, the reaction mixture was incubated for 10 min prior to spectroscopic measurements. Titration experiments were
repeated at least three times for each complex. Regression analyses
were performed, and lines giving an optimum correlation coefficient
were selected (23). Experimental errors were less than 20%.
Gel Filtration--
Gel filtrations were carried out using AKTA
liquid chromatography system equipped with Superdex 200 HR 10/30 column
(Amersham Biosciences). The buffer used for gel filtrations contained
50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, and 1 mM dithiothreitol.
Optical Absorption Spectra--
Optical absorption spectra of
Fe(III), Fe(II), Fe(II)-CO, Fe(II)-NO and Fe(II)-O2
complexes of Ec DOS are shown in Fig.
1 and summarized in Table
I. The Soret and visible absorption peaks of Fe(III), Fe(II), and Fe(II)-CO complexes of Ec DOS were
essentially the same as those of the isolated Ec DOS PAS
domain (18). The spectra of the Fe(III) and Fe(II) complexes of
Ec DOS are typical of six-coordinate low spin complexes,
which is different from those of the related enzyme, FixL, in which
both Fe(III) and Fe(II) complexes are penta-coordinate high spin
complexes (13, 14, 18). Fe(III) Ec DOS has a sharp Soret
band at 417 nm and distinct
In order to identify the axial ligands, the effect of modulating pH on
the absorption bands was examined. When the pH of the wild type
solution was varied from neutral to acidic, heavy precipitates were
formed. On the other hand, isolated Ec DOS PAS domain was stable but was insensitive to changes in pH from 5 to 9.5. Clear decreases in the intensity of the absorption spectra of the PAS domain
were observed outside this range (not shown). Denaturation rather than
axial ligand substitution is probably responsible for these effects.
CD, Fluorescence, and Redox Potential--
From the ultraviolet
region of the CD spectrum (Fig.
2A), the
Ec DOS has 16 Trp residues, whereas Ec DOS PAS
has 2 Trp residues. The fluorescence bands attributable to Trp are
located at 336 and 333 nm (excitation at 285 nm) for Ec DOS
and the isolated Ec DOS PAS domain, respectively (Fig.
2C). The fluorescence intensity of Ec DOS PAS was
half that of Ec DOS.
Electrochemical titrations were conducted for the isolated
Ec DOS PAS domain. The one-electron midpoint potential of
the Ec DOS heme was determined from the absorbance change at
562 nm. Half saturation points were +70 ± 1 mV for reduction and
+63 ± 8 mV for oxidation (Fig. 3).
No marked difference between the titration patterns in the two
directions was observed. Therefore, the redox potential of heme in the
isolated Ec DOS PAS domain was estimated to be +67 mV
versus SHE (n = 0.90).
Mutations at His-77 and His-83--
In order to identify the heme
axial ligands of the Ec DOS PAS domain, site-directed
mutagenesis of both His residues in the isolated PAS domain (His-77 and
His-83) was conducted. As shown in Fig.
4, the His-83 The Ec DOS Protein Is a Tetramer and the Isolated PAS Domain Is a
Dimer--
Purified Ec DOS protein and the isolated
Ec DOS PAS domain protein were located at 93- and 19-kDa
molecular masses on SDS-PAGE gels (Fig.
5, A and B). These
molecular masses, including the His6 tag (about 2 kDa), are
close to values predicted from amino acid sequences of both proteins.
Gel filtration was conducted to determine the molecular mass of
Ec DOS (Fig. 5, C and D). The estimated molecular mass of Ec DOS was 320 kDa, suggesting
that it is a tetramer. The molecular mass of the PAS domain was
similarly estimated to be 30 kDa (data not shown), suggesting it is a
dimer, as previously thought, even though the estimated mass is smaller than the calculated value (18).
Ec DOS Is a cAMP Phosphodiesterase--
Since the amino acid
sequence of the catalytic domain of Ec DOS is homologous to
that of AxPDEA1, cAMP or cGMP are the most probable substrates of
Ec DOS. Ant-cAMP was assayed for activity with Ec
DOS. Only the Fe(II) complex of Ec DOS had
enzyme-dependent PDE activity with cAMP (Fig.
6). We did not see detectable activity with the Fe(II)-O2 complex of Ec DOS perhaps due
to its very low turnover number or its instability. The
Fe(II)-O2 complex steadily decomposed to the Fe(III)
complex as mentioned above, which had no PDE activity.
The activity of cGMP with Fe(II) Ec DOS was marginal.
Bis(p-nitrophenyl) phosphate and p-nitrophenyl
phosphate, universal substrates for many PDEs, were not substrates for
Ec DOS.
The optimum reaction temperature was found to be between 30 and
40 °C. Thus, all catalytic activities were obtained at 37 °C in
the present study. The pH optimum for catalysis was found to be around
pH 8.5 (not shown). It was reported that divalent metal cations such as
Mn2+, Co2+, Ni2+, and
Zn2+ can support PDE activity, whereas Ca2+ is
virtually ineffective (21). Mg2+ was the most effective
dication for supporting catalysis with Ec DOS;
Ca2+ and Mn2+ were much less effective, and
Zn2+ was ineffective (not shown).
Addition of 1 mM dithiothreitol to the catalytic solution
enhanced the activity by 30%. This may be due to reduction of
interprotein disulfide bonds, causing a decrease in the amount of
aggregated enzyme, as indicated by gel filtration chromatography (not
shown). The turnover number of the enzyme under optimum conditions was 0.15 µmol/min/µmol of heme.
Inhibition Studies--
In order to confirm that the Fe(II) heme
regulates catalysis, CO and NO were added to the catalytic solution. As
shown in Fig. 7, CO and NO markedly
inhibited turnover. The IC50 values of CO and NO are about
1 µM or less. Dissociation constants for CO and NO were
about 0.3 and 0.5 µM, respectively, for Fe(II) Ec DOS, estimated by optical absorption titration (not
shown). Note that dissociation constants were calculated from the free CO or NO concentration, whereas the IC50 value reflects
total CO or NO concentration.
PDE inhibitors were examined to confirm that catalysis with
Ec DOS is PDE activity. Etazolate hydrochloride is a
selective inhibitor for cAMP-specific PDEs, whereas
3-isobutyl-1-methylxanthine is a nonspecific inhibitor for both cAMP
and cGMP PDEs (24). Both inhibitors strongly inhibited the
Ec DOS activity (Fig. 7C), reinforcing the
suggestion that Ec DOS really works as a PDE with cAMP.
The present work characterized the physicochemical and catalytic
properties of full-length Ec DOS and unequivocally
demonstrates for the first time that Ec DOS has PDE activity
with cAMP, which is regulated by the heme redox state.
Structural Characterization of Ec DOS
Optical Absorption Spectra--
From the absorption spectra, it is
clear that despite the close sequence similarity and expected
structural similarity between FixL and Ec DOS, the
environment of the heme iron in the two proteins is different (Table
I). The Fe(III) and Fe(II) FixL have high spin penta-coordinate heme
iron, whereas the spectra of Fe(III) and Fe(II) Ec DOS
full-length wild type are typical of hexa-coordinate low spin heme complexes.
The heme axial ligands of the Ec DOS PAS domain were thought
to be His and Met (18). Cytochrome b562 (25) and
cytochromes c (26), which have this type of coordination,
show clear pH-dependent spectral changes with apparent
pKa values between 9 and 6, caused by axial ligand
replacement. In contrast, the Ec DOS PAS domain appears
relatively stable to pH variation between 5 and 9.5, with no ligand
substitution. Therefore, either the heme environment is massively
different or there is a different pair of axial ligands to cytochrome
b562 and cytochromes c.
CD, Fluorescence, and Redox Potential--
The
The Soret CD band of Ec DOS is located only on the plus
side. This is in contrast with those of cytochrome
b562 (25) or cytochrome c (28) that
have bands on the minus side and both sides, respectively. The Soret CD
band of Ec DOS shifted from 421 to 425 nm concomitant with
the intensity increase upon reduction, suggesting that heme environment
was changed by reduction.
Fluorescence spectra with excitation at 280 nm are generally due to Tyr
and Trp. Ec DOS and the isolated Ec DOS PAS
domain contain 12 and 2 Trps, respectively. The fluorescence
intensities of Ec DOS and Ec DOS PAS appear to
correspond with the respective Trp numbers (Fig. 2C).
Solvated Trp residues should have a fluorescence maximum in the
vicinity of 350 nm, whereas those buried inside the hydrophobic core of
the molecule usually have a much lower wavelength (29, 30). The
fluorescence bands of Ec DOS and the isolated Ec
DOS PAS domain are located at 336 and 333 nm, respectively, whereas
both of the unfolded proteins (in the presence of 8 M urea)
are located at 349 nm.2 Taken
together, it appears that the Trp residues responsible for the
fluorescence band must be buried inside the protein or located in a
more hydrophobic environment. The reduction of Ec DOS heme
did not affect the peak position. This suggests that the environment of
these Trp residues was not changed by heme reduction.
The redox potential of Ec DOS was estimated to be +67 mV
versus SHE. From this value, it appears that the
Ec DOS Fe(III) complex is relatively stable compared with
the electron-transferring hemoproteins, cytochrome c (+260
mV versus SHE) (2, 26). However, Ec DOS may be
reduced more easily than cytochrome b5 (+3 mV)
(31), sperm whale myoglobin (+59 mV) (32), and microsomal P450 ( Axial Heme Ligands of Ec DOS--
In the UV-visible absorption
spectra of these mutants, it was clear that His-77, but not His-83, is
one of the heme axial ligands in the Ec DOS PAS domain. On
the other hand, modeling the structure of the Ec DOS PAS
domain based on that of the FixL heme-binding PAS domain led us to
predict that only Met-95, a residue on the heme distal side, is capable
of coordinating to the heme (18).
Tetramer--
Many heme sensor proteins such as CooA, soluble
guanylate cyclase, and Hem-AT have been known to be homo- or
heterodimer. FixL is also a soluble dimeric protein (29). Therefore, it
is not surprising that the Ec DOS PAS domain is dimeric
(18). However, it is interesting to note that Ec DOS itself
is tetrameric, although it has not been reported whether the
corresponding PDE with the similar PAS domain is dimeric or tetrameric
(24). Dimeric protein-protein interactions would regulate catalytic
function cross-wise and/or often regulate subcellular distribution (22,
24).
Catalytic Characterization
Catalytic Studies--
Ec DOS had PDE activity with cAMP but not
with cGMP, bis(p-nitrophenyl) phosphate, or
p-nitrophenyl phosphate. The latter two compounds are
universal substrates for many PDEs (20, 21, 24). Therefore, it appears
that Ec DOS has a high substrate specificity for cAMP. PDE
inhibitors, etazolate, hydrochloride, and 3-isobutyl-1-methylxanthine,
were antagonists for cAMP. The present results strongly support the
idea that Ec DOS is actually a PDE and has high specificity
for cAMP.
cAMP, synthesized by adenylate cyclase, is a very important cellular
mediator for regulation of catabolism and for E. coli cell
division. Therefore, Ec DOS may be involved in intravital signal transduction in E. coli. Imamura et al.
(30) reported a gene cording cAMP PDE which regulates intracellular
cAMP levels. We could not see a significant homology in amino acid
sequence between Ec DOS and their cAMP PDE. Although we do
not know what kind of cellular response is affected by cAMP level
regulation by Ec DOS PDE, it is very interesting it could be
directly controlled by the redox state of the cell. In addition, we
should note that Ec DOS has the highest homology to AxPDEA1.
The PDE activity obtained for Ec DOS (0.15 min
The relationship between heme redox state and activity was
investigated, and it appears that the Fe(II) form of Ec DOS
was active, whereas the Fe(III) form was not. If Ec DOS is
an O2 sensor enzyme, then it is implied that binding of
O2 to the ferrous heme itself or oxidation of heme might
cause the structural change necessary to regulate PDE activity under
normoxic conditions. This proposed mechanism is very similar to that
for FixL activation under hypoxic conditions. On the other hand, the
Fe(II)-O2 complex of Ec DOS was oxidized to
Fe(III) heme with a half-life 60 min. This rate is faster than that of
AxPDEA1 where a half-life was more than 12 h (19). When E. coli cells were crushed and centrifuged, optical absorption
spectrum of an Fe(II) complex at 428 nm was observed in the supernatant
solution at the initial stage. The spectrum of the Fe(II) complex
slowly converts to that of an Fe(III) complex at 417 nm. This suggests
that there is a possibility that the deoxy Fe(II) complex is the native form.
The catalytic domain of most PDEs constitutes the core sequence
including consensus metal-binding domains (Zn2+ and
Mg2+) related to those of metal-ion phosphohydrolases (24).
Although we have shown that Ec DOS requires Mg2+
for activity, it does not have the signature motif
HD(X2)H(X4)N, common to
many PDEs that is the sequence of the consensus metal-binding site
(Zn2+ and Mg2+). However, a similar motif
HD(X2)H(X4) is present in
Ec DOS, which may perform this function.
CO and NO were found to coordinate to the Fe(II) heme immediately, and
it is thought that the coordination of CO or NO caused a conformational
change in the heme-bound PAS domain. This structural change may
transfer a signal to affect the catalytic domain and cause a decrease
in activity. A recent resonance Raman spectroscopic study of CO and NO
complexes of heme-bound PAS domains suggested that the heme environment
of Ec DOS PAS was unusual compared with those of
other PAS domains (34).
In summary, the present study unequivocally indicates for the first
time that Ec DOS has PDE activity with cAMP, which is regulated by the heme redox state.
-helix content was calculated as 53%
by CD spectroscopy. The redox potential of the heme was found to be +67
mV versus SHE. Mutation of His-77 of the isolated PAS
domain abolished heme binding, whereas mutation of His-83 did not,
suggesting that His-77 is one of the heme axial ligands. Ferrous, but
not ferric, Ec DOS had phosphodiesterase (PDE) activity of
nearly 0.15 min
1 with cAMP, which was optimal at pH 8.5 in the presence of Mg2+ and was strongly inhibited by CO,
NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral
changes indicated tight CO and NO bindings to the ferrous heme.
Therefore, the present study unequivocally indicates for the first time
that Ec DOS exhibits PDE activity with cAMP and that this
is regulated by the heme redox state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside and 0.45 mM 
-aminolevulinic acid were added to the culture
medium, and further incubation with mild shaking at 25 °C was
continued for another 24 h. After incubation, the E. coli cells were harvested and crushed in buffer A (50 mM sodium phosphate buffer (pH 7.8), 10% glycerol)
containing aprotinin (final concentration 2 µg/ml), leupeptin (final
concentration 2 µg/ml), pepstatin (final concentration 2 mg/ml),
phenylmethylsulfonyl fluoride (final concentration 1 mM),
and 2-mercaptoethanol (final concentration 1 mM) by an
Ultrasonic Disrupter UD-201 (Tomy Co., Ltd., Tokyo, Japan). After
centrifugation at 100,000 × g for 30 min at 4 °C,
(NH4)2SO4 was added to the supernatant to precipitate the proteins. Ec DOS and the
isolated Ec DOS-PAS domain were collected from 0 to 50% and
30-70% (NH4)2SO4 solutions by
centrifugation at 10,000 rpm, respectively. The precipitates were
resolved in buffer A. The solution was passed though a Sephadex G-25
column (Amersham Biosciences), which was equilibrated with buffer
A, to remove (NH4)2SO4. The eluted
solution was then applied to a Ni-NTA-agarose column (Qiagen, Hilden,
Germany) pre-equilibrated with buffer A containing 20 mM
imidazole. The column was washed with buffer A containing 50 mM imidazole, and the protein was eluted with buffer A
containing 120 mM imidazole. The fractions containing the
Ec DOS protein were pooled and concentrated. Before using
the protein, imidazole was removed on a Sephadex G-25 gel filtration
column. For spectral experiments, DOS-PAS protein was further purified
by a gel filtration column of Sephadex G-75 (16 × 60 cm)
pre-equilibrated with 50 mM Tris-HCl buffer. Finally the
purified protein was quickly frozen in liquid N2 and stored at 
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and -bands in the 500-600 nm
region, whereas Fe(III) FixL has a broad blue-shifted Soret band at 396 nm and a broad band at around 500 nm, typical of a penta-coordinate
complex. The spectra of Fe(II) Ec DOS has a Soret band at
428 nm and well resolved - and -bands in the visible region, with
the -band (563 nm) being more intense than the -band (532 nm),
similar to those of AxPDEA1, whereas Fe(II) FixL has a Soret absorption
band at 437 nm and a broad band at around 556 nm, typical of a high
spin complex (13, 14, 18). The absorption peak of the Fe(II)-NO complex
of the Ec DOS was located at 423 nm. The
Fe(II)-O2 complex of Ec DOS was formed by adding
saturated O2 solution to the Fe(II) complex. This had an
absorption at 417 nm, similar to that of the isolated PAS domain (18).
The Fe(II)-O2 complex was unstable and gradually changed
the Fe(III) complex. Autoxidation of the Ec DOS
Fe(II)-O2 complex to the Fe(III) complex as monitored at 579 nm was composed of a single phase with rate constant about 1.5 × 102 min1.
View larger version (21K):
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Fig. 1.
Optical absorption spectra of Fe(III) ( 
),
Fe(II) (- - -), and Fe(II)-CO (···) (A) and
Fe(II)-NO () and Fe(II)-O2
(···) (B) complexes of Ec
DOS.
Optical absorption maxima of Ec DOS, AxPDEA1, and FixL; all of them
have heme-bound PAS domain (nm)
-helix content of
Ec DOS was estimated as 54% (CONTINLL program; 53% by the
SELCON3 program; 56% by the CDSSTR program). -Sheet content was
estimated to be about 10%. Reduction of the heme iron did not change
the CD spectrum in the ultraviolet region. The Soret CD band of the
resting Fe(III) complex was located at 421 nm on the plus side with
= 8 × 104 M1
cm1 (Fig. 2B). Reduction by sodium dithionite
shifted the band position to 425 nm and markedly increased the CD
intensity up to = 2 × 105
M1 cm1. The Fe(II)-CO complex
had a Soret band at 431 nm on the plus side with = 1 × 105 M1 cm1.
View larger version (18K):
[in a new window]
Fig. 2.
CD spectra of the UV (A) and
Soret (B) regions and fluorescence spectra
(C) of the near UV region of Ec
DOS. A, the solid line is the
experimental value, and the dotted line is estimated using
the CONTINLL program. B, Fe(III), Fe(II), and Fe(II)-CO
complexes are shown by the solid, broken, and
dotted-broken lines, respectively. The Soret CD spectra of
Ec DOS and Ec DOS PAS were essentially the same.
C, fluorescence bands of Ec DOS and Ec
DOS PAS are shown by broken and solid lines,
respectively.
View larger version (14K):
[in a new window]
Fig. 3.
Reductive and oxidative titrations of the
Ec DOS PAS domain. The fraction of the ferrous
form was plotted as a function of potential. The solid lines
are theoretical Nernst curves for reduction (square,
n = 0.82) and oxidation (circle,
n = 0.97) with midpoint potentials of 63 and 70 mV
versus SHE, respectively.
Gly and His-83 Ala
mutants had tight heme-binding capabilities and showed typical heme
absorption maxima at 417 nm, similar to that of the wild type PAS
domain. In contrast, the His-77 Gly and His-77 Ala mutants had
only very small absorption bands at the Soret region, suggesting that
very little heme was bound to these proteins consistent with His-77
being one of the heme axial ligands.
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[in a new window]
Fig. 4.
Optical absorption spectra of Fe(III)
complexes of mutants at His-77 and His-83 of the isolated PAS
domain. a, wild type; b, His-83 Gly;
c, His-83 Ala; d, empty vector; e,
His-77 Gly; and f, His-77 Ala.
View larger version (51K):
[in a new window]
Fig. 5.
SDS-electrophoresis gels and a gel
filtration chromatography pattern for the Ec DOS and
the isolated PAS domain. A, SDS-PAGE of the full-length
wild type: molecular marker (lane 1), cell supernatant
(lane 2), (NH4)2SO4
precipitates (lane 3), Ni-NTA flow-through (lane
4), Ni-NTA 20 mM imidazole elution (lane
5), and Ni-NTA 50 mM imidazole elution (lane
6) Ni-NTA 120 mM imidazole elution (lane
7); B, SDS-PAGE of the PAS domain: molecular marker
(lane 1), cell supernatant (lane 2), Ni-NTA
flow-through (lane 3), Ni-NTA 20 mM imidazole
elution (lane 4), Ni-NTA 50 mM imidazole elution
(lane 5), and Ni-NTA 120 mM imidazole elution
(lane 6); C, Superdex 200 gel filtration column
chromatography of the full-length wild type monitored by 400 nm;
D, correlation between elution volume of the gel filtration
and molecular weight to estimate the molecular mass of the full-length
wild type. Ec DOS is located at the far-right
end.
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Fig. 6.
PDE activities with cAMP of Fe(II)
(filled diamonds) and Fe(III) (open
squares) Ec DOS. Very little activity
was observed for cGMP with the Fe(II) enzyme (filled
circles). At least four experiments were conducted to obtain each
value, and experimental errors are less than 20%.
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Fig. 7.
Inhibitory effects of CO
(A), NO (B), and etazolate
hydrochloride and 3-isobutyl-1-methylxanthine (C) on
the Ec DOS activity. Etazolate hydrochloride is a
selective inhibitor for cAMP-specific PDE, whereas
3-isobutyl-1-methylxanthine is a nonspecific inhibitor for both cAMP
and cGMP PDE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix content
(54%) of Ec DOS estimated from the UV CD band suggests that
it consists of more -helical structure than FixL (37%), but not
much as Mb (78%) (4). The secondary structure of Ec DOS
appears to be different from either FixL or Mb (27).
310 mV) (33).
1) was about 50-fold lower than that of AxPDEAI (19)
but close to the activity (0.13 min1) of the cAMP PDE
isolated from E. coli cells (30). The activity of
Ec DOS was saturated with increasing the concentrations of Ec DOS (Fig. 6). A kind of product-inhibition or unfavorable
protein-protein interactions to affect the activity appear to exist,
but detailed mechanistic studies remain to be conducted.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of
Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Tel.: 81-22-217-5604 or 5605; Fax: 81-22-217-5604 or 5664; E-mail:
shimizu@tagen.tohoku.ac.jp.
Published, JBC Papers in Press, April 22, 2002, DOI 10.1074/jbc.M202738200
2 S. Hirata, T. Matsui, I. Sagami, and T. Shimizu, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CooA, a CO-sensing protein of R. rubrum; FixL, an oxygen sensor heme protein of R. meliloti; Ec DOS, full-length wild type of a direct oxygen sensor obtained from E. coli; PDE, phosphodiesterase; AxPDEA1, a phosphodiesterase A1 protein of A. xylinum; ant-cAMP, adenosine 3',5'-cyclic monophosphate, 2'-o-anthraniloyl; ant-cGMP, guanosine 3',5'-cyclic monophosphate, 2'-o-anthraniloyl; etazolate, 1-ethyl-4-[(1-methylethylidene) hydrazino]-1H-pyrazolo[3,4-b]pyridinde-5-carboxylic acid ethyl ester; SHE, standard hydrogen electrode; Ni-NTA, nickel-nitrilotriacetic acid.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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