SDS⁄ PAGE analysis of recombinant NOXtp revealed a molecular mass of approximately 50 kDa, which is close to that of the purified protein from T.. NOXtp is an FAD-dependent NADH and NADPH
Trang 1Hexameric ring structure of a thermophilic archaeon
Baolei Jia1,*, Seong-Cheol Park1,*,, Sangmin Lee1, Bang P Pham1, Rui Yu1, Thuy L Le1, Sang Woo Han2,3, Jae-Kyung Yang4, Myung-Suk Choi4, Wolfgang Baumeister5 and Gang-Won Cheong1,3
1 Division of Applied Life Sciences (BK21 Program), Gyeongsang National University, Jinju, Korea
2 Department of Chemistry, Research Institute of Natural Science, Gyeongsang National University, Jinju, Korea
3 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea
4 Division of Environmental Forest Science and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea
5 Department of Molecular Structural Biology, Max-Planck-Institute for Biochemistry, Martinsried, Germany
Thermococcus profundus is a thermophilic anaerobic
archaeon belonging to the Thermococcaceae family
that also includes Thermococcus kodakaraensis KOD1,
a model thermophilic organism whose whole genome
sequence has been reported [1] As an anaerobe living
in deep-vent environments, it seems likely that
Thermo-coccus encounters high levels of oxygen stress in the
water surrounding the vent [2] In anaerobes,
flavin-dependent NAD(P)H oxidases play an important role
protecting organisms from oxidative stress [3]
NADH oxidase (NOX) is a member of the flavo-protein disulfide reductase family that catalyzes the pyridine-nucleotide-dependent reduction of various substrates, including O2, H2O2 and thioredoxin [4] There are two types of NOX: those that catalyze the two-electron reduction of O2 to H2O2 and those that catalyze the four-electron reduction of O2 to H2O [5] The physiological role of NOX is diverse, depending
on its substrates and products in different organisms
In anaerobic mesophiles, NOX enzymes, such as those
Keywords
electron microscopy; H2O-producing;
hexameric ring structure; NADH oxidase;
thermophilic archaeon
Correspondence
G.-W Cheong, Division of Applied Life
Sciences, Gyeongsang National University,
Jinju 660-701, Korea
Fax: +82 55 752 7062
Tel: +82 55 751 5962
E-mail: gwcheong@gnu.ac.kr
Present address
Research Center for Proteineous Materials,
Chosun University, Kwangju 501-759, Korea
*These authors contributed equally to this
work
(Received 7 August 2008, revised 31 August
2008, accepted 3 September 2008)
doi:10.1111/j.1742-4658.2008.06665.x
An NADH oxidase (NOX) was cloned from the genome of Thermococ-cus profundus (NOXtp) by genome walking, and the encoded protein was purified to homogeneity after expression in Escherichia coli Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 49 kDa that exists as a hexamer with a native molecular mass of 300 kDa A ring-shaped hexameric form was revealed by electron microscopic and image processing analyses NOXtp catalyzed the oxidiza-tion of NADH and NADPH and predominantly converted O2 to H2O, but not to H2O2, as in the case of most other NOX enzymess To our knowl-edge, this is the first example of a NOX that can produce H2O predomi-nantly in a thermophilic organism As an enzyme with two cysteine residues, NOXtp contains a cysteinyl redox center at Cys45 in addition to FAD Mutant analysis suggests that Cys45 in NOXtp plays a key role in the four-electron reduction of O2 to H2O, but not in the two-electron reduction of O2to H2O2
Abbreviations
CoADR, coenzyme A disulfide reductase; GR, glutathione reductase; Nbs 2 , 5,5¢-dithiobis-(2-nitrobenzoic acid); NOX, NADH oxidase
(EC 1.6.99.3); NOXtp, Thermococcus profundus NADH oxidase.
Trang 2of Clostridium aminovalericum [6], Enterococcus
(Strep-tococcus) and Lactococcus [3], are considered to be
important enzymes in protecting against oxidative
stress and in regenerating oxidized pyridine
nucleo-tides through their capacity to reduce O2 to H2O
without the formation of harmful reactive oxygen
spe-cies Some NOX proteins have also been purified and
studied in (hyper)thermophilic organisms NOX from
Archaeoglobus fulgidus may be involved in electron
transfer in sulfate respiration [7] An H2O2-forming
NOX functions as an alkyl hydroperoxide reductase in
Amphibacillus xylanus [8] Some NOX enzymes, such
as those of Pyrococcus furiosus [9] and Thermotoga
maritima [10], have been proposed to protect
anaer-obes from oxidative stress In (hyper)thermophiles,
the roles of some NOX enzymes remain to be
elucidated [11]
NADH oxidase varies with the organism; however,
these proteins generally share similar secondary
struc-tural folding [4,12] An NOX from Thermus
thermophi-lus is a homodimer as determined by X-ray
crystallography [13] Gel filtration chromatography
indicated that NADH:flavin oxidoreductase from
Eubacterium is composed of three identical subunits
[14]; NOX in Clostridium thermohydrosulfuricum is
probably made up of six subunits, as demonstrated by
gel filtration [15] In contrast, a heterogeneous NOX
from Eubacterium ramulus is proposed to have an a8b4
assembly, as revealed by gel filtration and PAGE [16]
Two NOX enzymes from the Thermococcaceae
fam-ily have been described One is a novel enzyme in
P furiosus that produces both H2O2 (77%) and H2O
(23%) [9] The other NOX, in Pyrococcus horikoshii
OT3, may function as a CoA disulfide reductase
(CoA-DR) [17]; however, the function and structure of NOX
in Thermococcus, a genus of the Thermococcaceae
fam-ily, has not been clarified In this study, we have
cloned, overexpressed and purified a NOX that is
com-posed of two cysteine moieties from T profundus We
report its biochemical characterization and structure,
and also used mutants to analyze its catalytic
mecha-nism
Results
Cloning and sequencing of the nox gene from
T profundus
In order to clone the T profundus nox gene (NOXtp),
we utilized a PCR-based DNA-walking method using
the ClonTech genome-walker cloning kit, as described
in Experimental procedures; the resulting DNA
sequence comprised an ORF of 1329 bp, predicting a
protein composed of 442 amino acids with a molecular mass of 48 611 Da Figure 1 shows the nucleotide sequence of NOXtp and its flanking regions, together with the translated amino acid sequence The 5¢-flank-ing region of NOXtp contained a putative Archaea promoter with a TATA box and ribosome-binding site The 3¢-flanking region did not match with other Archaea genes, as judged by homolog searches in the NCBI database Unlike NOX, which has only one conserved cysteine residue (Cys45) in its N-terminus [4], the amino acid composition of NOXtp revealed the presence of two cysteine residues, Cys45 and Cys122 Additionally, two conserved cofactor-binding domains were also identified in NOXtp One was a FAD-binding domain containing the AMP-binding and FMN-binding motifs observed in enzymes belong-ing to the glutathione reductase (GR) family [18] The other domain was a glycine-rich NAD-binding motif located between the AMP-binding and FMN-binding motifs (two FAD-binding domains) (Fig 1) We pro-pose that NOXtp belongs to the GR family, because
of the high sequence identity of the cofactor-binding domains described above
Multiple sequence alignment (Fig S1) revealed that Cys45 is located at a similar position to that of the cysteine residue in the conserved active site of NOX from P horikoshii (also called CoADR) [17] and NOX and NADH peroxidase from Enterococcus faecalis [19,20] Sequence analysis by clustal w showed that NOXtp shared a significant level of identity with NOX (CoADR) from P horikoshii (80%) [17], NADH per-oxidase from E faecalis (28%) [20], and NOX from
P furiosus (36%) [9], Lactococcus lactis (30%) [21], Lactococcus sanfranciscensis (26%) [11] and E faecalis (27%) [19] (Fig S1) These proteins are generally com-posed of two identical subunits related by two-fold symmetry Each subunit can be divided into a C-termi-nal dimerization domain and an N-termiC-termi-nal pyridine nucleotide disulfide oxidoreductase domain, which is actually a small NADH-binding domain with a large FAD-binding domain [4,12] NOXtp has similar primary structure architecture to these proteins as determined by NCBI protein blast analysis
Purification of native and recombinant NOXtp
In order to understand the oxygen detoxification mecha-nism of anaerobic microbes, we purified NOXtp from
T profundus by several chromatographic methods The purified protein revealed a subunit with a molecular mass of approximately 50 kDa (Fig S2) The N-termi-nal amino acid sequence of purified NOXtp from T pro-funduswas determined to be MERKRVVIIGGGAAG,
Trang 3which is highly similar to that of NOX in T
kodakaren-sisKOD1, P furiosus, Pyrococcus abyssi and P
horiko-shiiOT3, belonging to the pyridine nucleotide disulfide
oxidoreductase family The purification of recombinant
NOXtp from Escherichia coli was performed by ion
exchange chromatography as described in Experimental
procedures SDS⁄ PAGE analysis of recombinant
NOXtp revealed a molecular mass of approximately
50 kDa, which is close to that of the purified protein
from T profundus (Fig S2) However, gel filtration
analysis under nondenaturing conditions showed that
the purified NOXtp had a molecular mass of
approximately 300 kDa (Fig 2) These results indicated
that NOXtp is a hexamer of 50 kDa subunits, in
contrast to NOX proteins from thermophilic archaeans,
which have been reported to be dimers or tetramers
[13,17]
Structure of NOXtp Gel filtration analysis under nondenaturing conditions revealed that purified NOXtp has a molecular mass
of 300 kDa, corresponding to a hexamer with
50 kDa subunits (Fig 2) This structure is different from that of other homologous NOX proteins, which consist of dimers or tetramers as revealed by X-ray crystallographic studies [12,13,22] In order to clarify the oligomeric structure of NOXtp, we performed electron microscopy using purified NOXtp The elec-tron micrographs of the negatively stained NOXtp oligomers showed a uniform distribution of the ring-shaped structure in the top-view orientation (Fig 3A) In total, 939 well-stained particles were translationally aligned, and were subjected to multi-variate statistical analysis [23] The eigenimages
Fig 1 Nucleotide sequence of the noxtp
gene and predicted amino acid sequence
of the gene product from
Thermo-coccus profundus The putative TATA-box
and ribosome-binding site are underlined
and in bold letters, respectively The
resi-dues involved in FAD binding are shadowed
in gray The NAD-binding site is boxed The
cysteine residues are in bold italic.
Trang 4obtained from the translationally, but not
rotation-ally, aligned images revealed a six-fold rotational
symmetry (Fig 3Ba) Using the 10 most significant
eigenvectors, nine classes were discriminated on the basis of similarity of features after rotational align-ment without symmetrization Most class averages showed a star-shaped structure with six-fold symme-try with heavy stain accumulation in its center (Fig 3Bb) In particular, the two class averages shown in panels 3 and 6 of Fig 3Bb exhibited an obvious deviation from the star-like structure This could result from incomplete stain embedding of the particle or from an unintentional inclination during preparation or microscopy In order to analyze further the rotational symmetry of the top-on-view images, the same dataset was separated into many classes (10–30) using different eigenimages (10–20) The dataset was also aligned with an arbitrarily chosen reference and separated according to the simi-larity of features in the eigenimages The resulting class averages revealed no other statistically signifi-cant symmetry (data not shown) We found no
Fig 2 Gel filtration chromatography profile of NOXtp purified from
Es coli The purified protein was subjected to Superdex-200 gel
fil-tration chromatography Absorbance was measured at 280 nm The
x-axis shows the elution time The standard proteins are ferritin
(440 kDa), catalase (232 kDa), albumin (67 kDa) and ovalbumin
(43 kDa).
A
B
a
b
Fig 3 Electron micrograph and structural analysis of NOXtp (A) Purified NOXtp was absorbed onto the grids as described in Experimental procedures The electron micrograph of the protein was then obtained
by negative staining with 2% uranyl acetate (B) Multivariate statistical analysis of NOXtp (a) The average (AV) of 939 translationally, but not rotationally, aligned particles with end-on orientation and the 10 most signifi-cant eigenimages (numbers 1–10) are shown In (b), the nonsymmetrized class averages (numbers 1–9) were derived from rotationally aligned images using the 10 most significant eigenvectors The numerals shown in the top right corner of the class averages are the number of particles seen
in each class (C) The average of the side-on view of NOXtp (939 particles) (D) A sche-matic model for the assembly of NOXtp complexes The diameters of the cavity, middle ring and outer ring are 4, 15 and
19 nm, respectively.
Trang 5evidence for the existence of a NOXtp protein with
intrinsically lower symmetry, at least at the resolution
employed
The average of the 939 top-on views revealed a
star-shaped structure (Fig 3C), which contained a middle
region of high density with heavy stain accumulation
in its center The average view also revealed that the
density of the complex was not homogeneous; the
den-sity increased towards the middle, such as seen in the
valosine-containing protein-like ATPase from
Thermo-plasma acidophilum complex, which is composed of
two stacked ring structures of different diameters [24]
The upper (or middle) ring of the NOXtp complex has
a region that is denser than that of the outer ring,
indi-cating the presence of a cavity in the complex with a
width of approximately 4 nm The diameters of the
outer ring and the middle ring (Fig 3D) were
approxi-mately 19 and 15 nm, respectively These projected
images as well as the gel filtration analysis indicated
that NOXtp predominantly exhibits a hexameric
star-shaped structure, in contrast to the structure recently
reported by Kuzu et al [22], which suggested a
tetra-meric structure for NOX from Lactobacilluus brevis
NOXtp is an FAD-dependent NADH and NADPH
oxidase
On the basis of the amino acid sequence, NOXtp
con-tains two FAD-binding domains The isoalloxazine
ring system in FAD has been suggested to induce light
absorbance in the UV and visible spectral range, giving
rise to the yellow appearance of flavin and
flavopro-teins [25] We performed light absorbance analysis to
confirm NOXtp binding to FAD Purified NOXtp from Es coli has absorption maxima at 378 and
456 nm, with a shoulder at 480 nm, which are charac-teristic spectral features of proteins with bound flavin cofactors (Fig 4A) The absorbance behavior also allowed the determination of the number of flavin mol-ecules bound per mole of NOXtp subunit [17,25,26] A stoichiometry of 0.7–0.9 mol FAD per mol NOXtp subunit was determined from the absorbance at
460 nm
As NOXtp contains FAD as a prosthetic group, apo-NOXtp was prepared by hydrophobic interaction chromatography under acidic conditions (pH 3.5) with saturated NaBr buffer [26,27], in order to
0 0.04 0.08 0.12 0.16 0.2
0 10 20 30 40 50 60 70 80 90 100
0 1 2 3 4 5 6 7 8
0 1 2 3 4 5 6 7 8
–1 )
A
B
C
D
Fig 4 Activity assays of NADH and NADPH oxidase (A) Visible
spectra of NOXtp (solid line), apo-NOXtp (dashed line) and the
C45A mutant (dotted line) The absorbance was measured in
50 mM sodium phosphate buffer (pH 7.2) at 25 C (B) FAD effect
on NAD(P)H oxidase activity An activity assay was performed as
described in Experimental procedures The solid line shows the
NADH oxidase activity of NOXtp purified from Es coli (h),
reconsti-tuted NOXtp (s), and apo-NOXtp (4) The dashed line shows the
NADPH oxidase activity of NOXtp from Es coli (h), reconstituted
NOXtp (s), and apo-NOXtp (4) (C) Optimal temperature of NAD(P)H
oxidase activity The assay was performed at the indicated
temper-atures in 50 mM potassium phosphate buffer (pH 7.2) NADH and
NADPH oxidase activity are shown by a solid line and a dashed
line, respectively The squares show the measured temperature
points (D) Optimal pH of NAD(P)H oxidase activity Different buffers
were used in this assay Sodium phosphate was used at pH 6.0,
6.6, 7.2 and 7.7; Hepes buffer and Tris buffer were used at pH 8.0
and 8.5; sodium borate buffer was used at pH 9.0 These buffers
were used at a concentration of 50 mM NADH and NADPH
oxi-dase activity are shown by a solid line and a dashed line,
respec-tively The squares show the measured pH points.
Trang 6confirm the function of FAD The absorption
spec-trum of apo-NOXtp did not show any significant
absorbance in the visible region, revealing that FAD
was indeed absent (Fig 4A) To determine whether
FAD was required for the enzymatic activity of
NOXtp, holoprotein and apoprotein activities were
assayed The NADPH oxidase activity of NOXtp
was also measured, as described previously for NOX
(CoADR) from P horikoshii [17] and NOX from
L sanfranciscensis [12], which show high similarity to
NOXtp and accept both NADH and NADPH as
cofactors The activity of the reconstituted enzyme,
which was accomplished by incubating equimolar
concentrations of apomonomers and FAD at room
temperature for 5 min [26], was also measured These
assays revealed that NADH oxidase activity was
slightly higher than that of NADPH oxidase, and
FAD significantly restored the oxidase activity of
apo-NOXtp (Fig 4B) These results clearly indicated
that NOXtp is an FAD-dependent NADH and
NADPH oxidase, in contrast to NOX enzymes from
other thermophilic archaeons, which only exhibit
activity towards NADH [9–11]
To further determine the function of NOXtp, the
steady-state kinetic parameters of NOXtp with either
NADH or NADPH as the reducing substrate were
measured at pH 7.2 NOXtp could catalyze NADH
and NADPH oxidization with kcat values of
6.2 ± 0.5⁄ s and 2.5 ± 0.3 ⁄ s, respectively The
steady-state kinetic parameters of NOXtp were similar to
those of NOX (CoADR) from P horikoshii (Table 1)
On the basis of the Km, both enzymes preferred
NADPH as the substrate for oxidase activity,
indicat-ing that NOX (CoADR) from P horikoshii and
NOX-tp belong to similar enzyme families The optimal
temperature for the NADH and NADPH oxidase
activity of NOXtp was near 70C (Fig 4C), which is
lower than the optimal growth temperature (80C) of
this organism The optimal pH was between 7.5 and
8.0 for both NADH and NADPH oxidase activity
(Fig 4D)
NOXtp preferentially produces H2O The product of O2 reduction is an important factor in evaluating the physiological function of NOXs [10] For instance, NOX from P furiosus, which may pro-tect anaerobic thermophiles against oxidative stress, can produce both H2O2 and H2O [9] In order to determine the product of the NAD(P)H oxidase activ-ity of NOXtp, reactions containing 100 lm NAD(P)H were performed [all NAD(P)H consumed] according to the published method [9], and H2O2 was quantified using a peroxi-DETECT kit from Sigma (St Louis,
MO, USA) When NADPH oxidation was performed
at 80 C, approximately 7% of the NADPH supplied was used to produce H2O2, and 2% of the NADH was recovered as H2O2 under the same conditions (Fig 5D) These results demonstrated that NOXtp produces predominantly H2O using NADH and NADPH as electron donors
Cys45 but not Cys122 functions as the nonflavin redox center
NADH oxidase in members of the Thermococcaceaee family, such as T kodakaraensis KOD1, P horrikoshii,
P abyssi and P furiosus, have only one conserved cys-teine residue, Cys45; however, the sequence of NOXtp (Fig 1) revealed that it contains two cysteine residues, Cys45 and Cys122 As cysteines are important residues for NOX enzyme activity, we replaced Cys45 and Cys122 with alanines to analyze the function of these two residues After purification using the same method
as that used for the wild-type enzyme, the number of cysteines in the three mutant enzymes (NOXtpC45A, NOXtpC122A and NOXtpC45A⁄ C122A) was exam-ined using Ellman’s method (Table 2) The single mutants, NOXtpC45A and NOXtpC122A, contained about one cysteine, and the double mutant contained
no cysteines These data confirmed the identity of the mutants and also indicated that the nonmutated cyste-ine remacyste-ined in its native state The visible absorption spectra showed that the three mutants contained tightly bound FAD (Fig 4A, NOXtpC45A only shown – the other two mutants produced similar absorbance spectra) Electron microscopy and native PAGE showed no significant difference between wild-type NOXtp and its mutants (Fig 5A,B) All of the data indicated that the disulfide bond was not respon-sible for hexameric oligomerization and that substitu-tion of Cys45 and Cys122 with alanine did not result
in major changes in NOXtp quaternary structure
In order to determine the catalytic mechanism of NOXtp, NAD(P)H oxidase assays were performed
Table 1 Steady-state kinetic parameters of NOXtp and NOX
(CoA-DR) from Pyrococcus horikoshii (50 mM potassium phosphate
buf-fer, pH 7.2, 75 C) Data shown are means of triplicate
determinations ± SD.
Parameter
NOXtp-NADH
oxidase
NOXtp-NADPH oxidase
CoADR-NADH oxidase
CoADR-NADPH oxidase
kcat(s)1) 6.2 ± 0.5 2.5 ± 0.3 8.2 a 2.0 a
a From reference [17].
Trang 7with the three mutants under the same conditions as
used for the wild-type The results showed that the
C122A mutant had similar NADH and NADPH
oxi-dase activity to that of the wild-type protein; however,
the C45A mutant and the double C45A⁄ C122A
mutant had < 10% of the NAD(P)H oxidase activity
of the wild-type protein (Fig 5C) These results are
similar to those obtained with a NOX from E faecalis,
where a serine substitution of its active site residue
Cys42 (C42S) resulted in approximately 3% of the
activity of the wild-type under the same conditions
[4,28] Considering these results, Cys45 may provide
the essential second redox center in addition to the
fla-vin We further examined the products of NOXtp and
its mutants NAD(P)H oxidation was allowed to go to
completion, and the amount of H2O2 formed in the
reaction was quantified using the peroxi-DETECT kit
The NOXtpC122A mutant produced a similar amount
of H2O as the wild-type under the same conditions
and with the same substrates (Fig 5D) Oxidation of
NADH and NADPH by NOXtpC45A and
NOX-tpC45A⁄ C122A led to the formation of about one
equivalent of H2O2 (Fig 5D), demonstrating that
H2O2 production by these two mutants is stoichiome-tric with NADH and NADPH oxidation The activity and product assays using the wild-type and mutants clearly demonstrated that Cys45 participates in the direct four-electron reduction of O2 to H2O, and the Cys45 mutation alters the reaction to produce H2O2 instead of H2O
Discussion
In this study, we have demonstrated that NOXtp has a hexameric ring-shaped structure Gel filtration under nondenaturing conditions revealed that NOXtp is com-posed of six subunits Moreover, upon electron micro-scopic analysis, NOXtp was found to predominantly exhibit a hexameric structure that contained a middle region of high density with heavy stain accumulation
in its center However, the crystal structure of NOX from L sanfranciscensis revealed a dimeric form with
an N-terminal oxidoreductase domain and a C-termi-nal dimerization domain [12] NPX from Streptococ-cus faecalis, catalyzing the conversion of H2O2 to
H2O, was reported to be a homotetrameric structure [29] These two mesophilic proteins show different types of subunit oligomerization and low sequence identity (Fig S1), but each of their subunits shows high structural similarity and their folding patterns are similar to that of GR [12,29] In contrast, NOX from Thermoanaerobium brockii was found to have a hexa-meric quaternary structure by gel filtration [15] Elec-tron microscopic analysis has revealed that NOXtp has
a hexameric ring-shaped structure composed of two
0 1 2 3 4 5 6 7 8
d c b
a a b c d
100 nm
100 nm
0 20 40 60 80 100
H 2
O 2
d c b
a a b c d
669 kDa 440
67
140 232
D
Fig 5 Comparisons of activity, products and structures between NOXtp and the mutants (A) Electron micrographs of NOXtp (a), NOX-tpC45A (b), NOXtpC122A (c) and NOXNOX-tpC45A ⁄ C122A (d) The bar represents 100 nm (B) Native PAGE of wild-type NOXtp and its mutants Lanes 1–4 correspond to (a), (b), (c) and (d) in (A), respectively; lane 5 is the molecular weight marker The lower part shows the correspond-ing proteins determined by SDS ⁄ PAGE (C) Specific activity of wild-type NOXtp (a), NOXtpC45A (b), NOXtpC122A (c) and NOX-tpC45A ⁄ C122A (d) with NADH (bar 1) and NADPH (bar 2) as substrates (D) The amount of H 2 O 2 produced by NOXtp (a), NOXtpC45A (b), NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d) when 100 lM NADH (bar 1) or 100 lM NADPH (bar 2) was oxidized.
Table 2 Determination of the sulfhydryl contents of wild-type and
mutant NOXtp using Ellman’s reagent Data shown are means of
triplicate determinations ± SD.
Protein
No cysteines per protein
Trang 8stacked rings of different diameters (19 and 15 nm
respectively) that encompass a central opening; this is
the first hexameric NOX determined by electron
microscopy Significantly, this structural feature of
NOXtp is highly similar to that of valosine-containing
protein-like ATPase from Th acidophilum, an archaeal
member of the AAA family (ATPases associated with
a variety of cellular activities) [24] In addition, the
structure of the cysteine mutants, NOXtpC45A,
NOX-tpC122A and NOXtpC45A⁄ C122A, was the same as
that of the wild-type Thus, it appears that a disulfide
bond does not participate in the oligomerization and
quaternary structure of NOXtp
NADH oxidase catalyzes the transfer of electrons
from reduced pyridine nucleotides to O2 [2,4] Here we
have demonstrated that NOXtp can efficiently reduce
O2 to produce H2O using both NADH and NADPH
as electron donors In addition, the activity and
prod-uct assays of the wild-type and mutants showed that
Cys45 is the active site residue and that Cys122 does
not function in the NADH and NADPH oxidase
activity These results indicate that Cys45 participates
in the direct four-electron transfer reduction of O2 to
H2O, and that the Cys45 mutant alters the reduction
to produce H2O2 instead of H2O, similar to NOX in
E faecalis [28] NOX in E faecalis belongs to a group
of enzymes that use a cysteine sulfenic acid as the
non-flavin redox center These enzymes are found in
Enterococcusand Streptococcus, which are aerotolerant
anaerobes, where they play an important role in O2
tolerance [4] For example, H2O-forming
NOX-defi-cient mutants of Streptococcus pyogenes are unable to
grow under high-O2 conditions, revealing the
impor-tance of NOX-scavenging activity against harmful O2
[30] We therefore propose that NOXtp may
decom-pose O2in the anaerobe T profundus
The predominant production of H2O by NOXtp is
in contrast to the exclusive production of H2O2 by
most NOXtp homologs in thermophiles, such as NOX
in A fulgidus, Desulfovibrio gigas, Thermot maritima
and Thermoanaerobium brockii [10,11,15,31] Previously,
the production of H2O2 was considered to be the
dis-tinctive property of NOX proteins from thermophiles
[10,11], with the exception of NOX from P furiosus,
which produces both H2O2 (77%) and H2O (23%) [9]
To our knowledge, NOXtp is the first NOX to be
purified from thermophilic microorganisms that can
catalyze electron transfer from NADH and NADPH
to O2 and predominantly produce H2O NOXtp is
therefore better for removing O2 than other reported
O2-scavenging systems, which must employ
intermedi-ates to reduce H2O2 produced by NAD(P)H oxidases,
such as in D gigas, where rubredoxin and neelaredoxin
act as intermediates [31] As NOXtp and the
mesophil-ic enzymes that decompose injurious O2 belong to the same group (discussed above), and NOXtp reduces O2
to H2O directly, we propose that NOXtp may play an important role in O2 removal or aerobic tolerance in thermophilic anaerobes
Experimental procedures
Purification of NOXtp from T profundus
reported previously [32] After harvesting, the cells were dis-solved in 20 mm potassium phosphate buffer (pH 6.5),
and 10% glycerol (PMEDG buffer), and disrupted by soni-cation The homogenates were centrifuged at 10 000 g for
30 min The supernatant was loaded on a phosphocellulose column that had been equilibrated with PMEDG buffer After being washed completely, the proteins were eluted by
100, 200, 300, 400, 500 and 1000 mm NaCl in a stepwise gradient, and the eluates in 200 mm NaCl were dialyzed with 50 mm Tris buffer (pH 8.0) containing 400 mm NaCl The sample was then loaded on an amino-benzimide col-umn equilibrated with the same buffer Unabsorbed pro-teins on the resin were collected and dialyzed with PMEDG buffer, concentrated using a centricon (Millipore, Billerica,
checked by transmission electron microscopy The protein concentration was determined by the Bradford method, and BSA was used as standard
SDS/PAGE and N-terminal sequencing
elec-troblotted onto poly(vinylidene difluoride) membranes The visible band was excised and applied to a protein sequence analyzer (Korea Basic Science Institute, Daejeon, Korea)
Cloning of NOXtp from T profundus
Polymerase chain reaction experiments with T profundus genomic DNA as a template were performed using
TGC AT-3¢; N = A, G, C and T; Y = C and T; W = A and T) The sense primer was designed from the known N-terminal sequence, and the antisense primer was from the conserved C-terminal sequence of NOX The experi-ment using the two oligonucleotides afforded an amplificate
confirmed by sequencing The resulting sequence was used
Trang 9for subsequent cloning Full gene cloning of NOXtp was
per-formed using the Universal Genomewalker kit (ClonTech,
Mountain View, CA, USA) Briefly, the genomic DNA was
digested with EcoRV, DrabI, PvuII and SspI separately, and
ligated to the adaptor provided by the kit PCR was
per-formed with the adaptor primers (provided by the kit) and
GCT GTA AAT GCC GAG AT-3¢), which corresponded
to the known sequence detected by the degenerate primers
The PCR products were ligated into the pTOPO vector,
transformed, and sequenced
Expression and purification of NOXtp in Es coli
TGG AGAGGAAACGCGTTGTTAT-3¢; antisense primer,
5¢-CGCG AAGCTT TAAAACTTTAGAACCCTG-3¢) were
designed on the basis of the sequence of the Genomewalker
result (the underlined bases indicate the restriction enzyme
site) The PCR product and pET28-(a) were digested by
NcoI and HindIII and ligated The ligation product was
transformed into Es coli BL21(DE3) by electroporation
Finally, the recombinant vector (pENOXtp) was confirmed
by sequencing
Recombinant Es coli cells (2 L) were cultured in LB
iso-propyl-thio-b-d-galactoside for 4 h After harvesting, the
cells were resuspended in PMEDG buffer and disrupted by
sonication After centrifugation (3000 g, 30 min), the
denatured proteins were removed by centrifugation (3000 g,
30 min) The supernatants were loaded onto a
phosphocel-lulose column that had been equilibrated with the same
buffer After being washed completely, the proteins were
eluted with 200 mm NaCl The purified protein was
Mutagenesis
The primers used for the single cysteine to alanine mutants
were as follows: C45A, forward primer, 5¢-ACG GAA
TGG GTG AGC CAC GCT CCC GCC GGT ATC CCC
5¢-ACC CTC AAC TAC GTA GGG GAT ACC GGC GGG
for-ward primer, 5¢-CCG CAG GTT CCG GCG ATA GAG
GGC GCC CAC CTG GAA GGA GTA TTC ACA
GCA-3¢; and C122A reverse primer, 5¢-TGC TGT GAA TAC
TCC TTC CAG GTG GGC GCC CTC TAT CGC CGG AAC
CTG CGG-3¢ The PCR was performed using Pfu
polymer-ase (Takara, Kyoto, Japan), and the cycling parameters
for 12 min (12 cycles) After amplification, the PCR mixture was digested with DpnI and then transformed into Es coli BL21(DE3) by electroporation The mutants were confirmed
by DNA sequencing The double cysteine mutants were produced by the same method, except that pENOXtpC45A was used as the template and C122A primers were used for the amplification The mutant proteins were purified using the same method as used for wild-type purification
Gel filtration chromatography
column (Amersham Biotech, Piscataway, NJ, USA)
(232 kDa), albumin (67 kDa) and ovalbumin (43 kDa)
Apo-NOX preparation
The purified NOXtp from Es coli is a holoenzyme with FAD The protein was dialyzed with 100 mm phosphate
dith-iothreitol and 0.5 mm EDTA, and then loaded on the hydrophobic interaction chromatography column brated with the same buffer FAD was eluted with equili-bration buffer saturated with NaBr (pH 3.5) The column was balanced again with the equilibration buffer, and the apoprotein was eluted with 100 mm phosphate buffer [26,27] Eluates were dialyzed with the PMEDG buffer
Enzyme assays
The NADH or NADPH oxidase activity of the recombi-nant protein was examined by time-dependent removal of NAD(P)H in aerobic conditions The assays were per-formed in 50 mm sodium or potassium phosphate buffer (pH 7.2), 0.5 mm NAD(P)H and 100 mm NaCl at the indi-cated temperatures The reaction was started by adding NOXtp in the amounts indicated The rate of NAD(P)H consumption was measured by monitoring the decrease in
A340 nm One unit of activity was defined as the amount of enzyme catalyzing the oxidation of 1 lmol NADH per
(pH 7.2) and 0.5 mm NADH To measure kinetic parame-ters, reaction rates were measured at a series of NAD(P)H concentrations, and the rates at various substrate concen-trations were finally fitted by Lineweaver–Burk plots The parameters (with standard deviation) were determined by three separate experiments
Determination of the sulfhydryl content
The sulfhydryl contents were determined using Ellman’s reagent in anaerobic conditions according to a published
Trang 10method [17,33] After the proteins and
The sulfhydryl concentrations in these proteins were
deter-mined from a calibration curve created using known
con-centrations of standard l-cysteine solutions
H2O2detection
Briefly, the assay was performed in 50 mm sodium
phos-phate buffer (pH 7.2), 100 lmol NAD(P)H, 1 mm EDTA,
100 mm NaCl and 0.2 nmol NOXtp The reaction was
allowed to go to completion Reaction buffer (100 lL) was
adduct with xylenol orange, which is observed at 560 nm
NAD(P)H must be consumed completely
Electron microscopy and image processing
Purified NOX was applied to glow-discharged
carbon-coated copper grids After the proteins had been allowed to
absorb for 1–2 min, the grids were rinsed with droplets of
Electron micrographs were recorded with an FEI
120 kV
Light-optical diffractograms were used to select the
micrographs, to examine the defocus and to verify that no
drift or astigmatism was present Suitable areas were
a pixel size of 20 lm, corresponding to 0.38 nm at the
spec-imen level For image processing, the semper [34] and em
[35] software packages were used From digitized
individ-ual particles were extracted interactively These images were
aligned translationally and rotationally, using standard
cor-relation methods [36,37] An arbitrarily chosen reference
was used for the first cycle of alignment and averaging, and
the resulting average was used as a reference in the second
refinement cycle For analysis of the rotational symmetry of
top-on-view images, the individual images were aligned
translationally but not rotationally [38] These aligned
images were subjected to multivariate statistical analysis
[39] The resulting eigenimages represent all-important
structural features of the original dataset If the images had
different rotational symmetries in the original dataset, the
eigenimages would reveal the different symmetry axes
Moreover, these images can be distinguished and
subse-quently separated on the basis on eigenimages The
rota-tionally aligned images were classified on the basis of
eigenvector–eigenvalue data analysis, and subsequent aver-aging was performed for each class separately The average was finally symmetrized on the basis of angular correlation coefficients [40]
Acknowledgements
B Jia, S Lee, B P Pham, R Yu and T L Le were supported by scholarships from the Brain Korea21 project in 2008, Korea This work was supported by a grant from the MOST⁄ KOSEF to the Environmental Biotechnology National Core Research Center (grant
no R15-2003-012-01003-0), and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (grant no KRF-2007-521-C00241), to
G W Cheong
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