de Vos Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, the Netherlands Three NADH oxidase encoding genes noxA-1, noxB-1 and noxC were c
Trang 1Molecular characterization of H2O2-forming NADH oxidases
Serve´ W M Kengen, John van der Oost and Willem M de Vos
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, the Netherlands
Three NADH oxidase encoding genes noxA-1, noxB-1
and noxC were cloned from the genome of Archaeoglobus
fulgidus, expressed in Escherichia coli, and the gene products
were purified and characterized Expression of noxA-1 and
noxB-1resulted in active gene products of the expected size
The noxC gene was expressed as well but the protein
pro-duced showed no activity in the standard Nox assay
NoxA-1 and NoxB-NoxA-1 are both FAD-containing enzymes with
subunit molecular masses of 48 and 69 kDa, respectively
NoxA-1 exists predominantly as homodimer, NoxB-1 as
monomer NoxA-1 and NoxB-1 showed pH optimum of
8.0 and 6.5, with specific NADH oxidase activities of
5.8 UÆmg)1 and 4.1 UÆmg)1, respectively Both enzymes
were specific for NADH as electron donor, but with different
apparent Kmvalues (NoxA-1, 0.13 mM; NoxB-1, 0.011 mM)
The apparent Km values for oxygen differed significantly (NoxA-1, 0.06 mM; NoxB-1, 2.9 mM) In contrast with all mesophilic homologues, both enzymes were found to pro-duce predominantly H2O2instead of H2O Despite apparent similarities, NoxB-1 is essentially different from NoxA-1 Whereas NoxA-1 resembles typical H2O-producing Nox enzymes that are expected to have a role in oxidative stress defence, NoxB-1 belongs to a small group of enzymes that
is involved in catalysing the reduction of unsaturated acids and aldehydes, suggesting a role in fatty acid oxidation Moreover, NoxB-1 contains a ferredoxin-like motif, which
is absent in NoxA-1
Keywords: Archaeoglobus; flavoprotein; NADH oxidase; oxygen stress
Archaeoglobus fulgidusis a strictly anaerobic
hyperthermo-philic archaeon that has been isolated from marine
hydro-thermal environments as well as subsurface oil fields This
sulfate reducer can grow organoheterotrophically with
a variety of carbon sources, or lithoautotrophically on
hydrogen, thiosulfate and CO2[1] Besides its ability to grow
at extremely high temperatures, this organism is unusual in
that it is evolutionary unrelated to other sulfate reducers
Recently, the sequence of the entire genome of A fulgidus
was completed [2] The sequencing revealed the presence of
eight putative NADH oxidase genes, which were designated
noxA-1to noxA-5, noxB-1, noxB-2 and noxC, according to
their homology to other NADH oxidase encoding genes
NADH oxidases (EC 1.6.99.3) catalyse the two-electron
reduction of oxygen to peroxide or the four-electron
reduction of oxygen to water Although all so-called
NADH oxidases share the ability to reduce oxygen, their
physiological role may differ or is often not known
Moreover, some homologues have been shown not to
reduce oxygen and to catalyse somewhat different reactions, such as NADH peroxidase (EC 1.11.1.1) and disulfide reductase (EC 1.8.1.14) The noxA homologues from
A fulgiduscode for a group of typical H2O-forming NADH oxidases of 49 kDa, found in various prokaryotes, and including the well-studied NADH oxidase from Enterococ-cus faecalis[3] (Fig 1) This group also contains NADH peroxidases and coenzyme A disulfide reductases [4,5] The noxB homologues encode a small group of enzymes of
72 kDa, that are involved in the reduction of unsaturated acids and aldehydes, or whose function is not known In the presence of oxygen, they also produce H2O, except for the enzyme from Thermoanaerobacter brockii that forms H2O2 and some superoxide (O2)) [6] NoxC codes for a small 20-kDa protein, and it was designated as NADH oxidase, due to its homology to the NADH oxidases from the thermophilic bacteria Thermus aquaticus and Thermus thermophilus [7,8] Some putative NAD(P)H oxidoreduc-tases from thermophilic archaea also belong to this group (Fig 1)
The physiological role of the putative NADH oxidases in
A fulgidusis still enigmatic Common NADH oxidases of mesophilic origin are assumed to protect the cells from oxidative stress by reducing oxygen to water, without the formation of harmful reactive oxygen species Alternatively, NADH oxidases may recycle oxidized pyridine nucleotides during catabolism Moreover, some homologues were shown to have functions other than NADH oxidase (see above) Concerning the homologues from (hyper)thermo-philic species, only a few have been studied in more detail A recently purified NADH oxidase from A fulgidus (NoxA-2) was proposed to be involved in electron transfer reactions
Correspondence to S W M Kengen, Laboratory of Microbiology,
Hesselink van Suchtelenweg 4, 6703 CT Wageningen, the Netherlands.
Fax: +31 317 483829, Tel.: +31 317 483748,
E-mail: serve.kengen@wur.nl
Abbreviations: NoxA-1, NADH oxidase A-1; NoxB-1, NADH
oxidase B-1; NoxC, NADH oxidase C; DCPIP, 2,6
dichlorophenol-indophenol; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid).
Enzymes: NADH oxidases (EC 1.6.99.3); NADH peroxidase
(EC 1.11.1.1); disulfide reductase (1.8.1.14).
(Received 28 February 2003, revised 25 April 2003,
accepted 14 May 2003)
Trang 2during sulfate respiration [9] The NADH oxidase of
Pyrococcus furiosus (Nox1; PF1430634) purified from an
overproducing Escherichia coli, was anticipated to play a
role in protection against oxidative stress [10] The function
of the NADH oxidase of T brockii is still unknown [6] In
several hyperthermophilic archaea and bacteria, other than
A fulgidus, various putative NADH oxidase genes have
been identified, whose function remains to be established
Independent of their physiological role, H2O2-producing
NADH oxidases may be applicable in biosensors, where
they act as mediator between NADH-forming
dehydrogen-ases and the electrode [11] Concerning this, enzymes from
(hyper)thermophiles may be superior to mesophilic
coun-terparts, because they generally exhibit a higher stability not
only with respect to temperature, but also towards chemical
denaturants, like detergents or organic solvents [12,13]
In order to analyse the biochemical properties, to unravel
their physiological role and to test the potential stability, the
noxA-1 gene, the noxB-1 gene and the noxC gene were
cloned and expressed in E coli and the overproduced
NADH oxidases were purified and characterized
Experimental procedures
Materials
3,3¢-Dimethoxybenzidine, coenzyme A (oxidized),
glutathi-one (oxidized), horseradish peroxidase and
2,3-dimethyl-1,4-naphthoquinone were from Sigma Chemie
Q-Sepharose, Superdex 200 HR 10/30, and Mono-Q HR
5/5 were from Amersham Pharmacia Biotech
Hydroxy-apatite (Bio-Gel HT), SDS/PAGE calibration proteins
(broad range), and the protein assay kit were from Bio-Rad
The pET9d expression vector was from Novagen Inc
E coli BL21(DE3) and Pfu DNA polymerase was from Stratagene A fulgidus (DSM 4304) was from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) Polysulfide was prepared by adding 12 g Na2S to 1.6 g elemental sulfur in 100 mL anoxic water [14] Cytochrome c was purified from the mesophilic Syntrophobacter fumaroxidans and was a gift of Frank de Bok (Wageningen University, the Netherlands) Cloning of the NADH oxidase genes
In the genome sequence of A fulgidus [2] various putative NADH oxidase genes have been identified, of which three were selected for further research (noxA-1, AF0254; noxB-1, AF0455; noxC:, AF0226) The following primer sets were designed to amplify the selected Nox open reading frames: for noxA-1 primer BG852 (5¢-CGCGTCATGAAGGTT GCAATTATAGGCGGT-3¢, sense) and primer BG853 (5¢-CGCGGGATCCCTACGGCAATCCGAGCTTC-3¢, antisense), with BspHI and BamHI restriction sites in bold; for noxB-1 primer BG854 (5¢-CGCGCCATGGCCAAG CTTTTCGAGCCAATCGAG-3¢, sense) and BG855 (5¢-CGCGGGATCCCTAAACCTTCAAAGCCAGAT-3¢, antisense), with restriction sites NcoI and BamHI in bold; for noxC primer BG831 (5¢-GCGCGTCATGATGGAAT GCCTTGACTTGCTGTTC-3¢, sense) and BG832 (5¢-CG CGCGGATCCTCACCATTTTTCGAAGTGCGTGAG-3¢, antisense), with BspHI and BamHI restriction sites in bold The 50 lL PCR reaction mixture contained 200 ng A ful-gidusSL-5 genomic DNA, isolated as described previously [15], 100 ng each primer, 0.3 mM dNTPs, Pfu polymerase buffer, and 2.5 U Pfu DNA polymerase and was subjected
to 35 cycles of amplification (15 s at 94C, 30 s at 50 C and 2 min at 68C) on a DNA Thermal Cycler (Perkin Elmer Cetus) The PCR product was digested with the appropriate enzymes and cloned into an NcoI/BamHI-digested pET9d vector, resulting in pWUR66, pWUR67, and pWUR68, for noxA-1, noxB-1 and noxC, respectively The plasmids were transformed into E coli TG1 and E coli BL21(kDE3) by heat-shock Sequence data were analysed using the computer programDNASTAR
Expression of the NADH oxidase genes inE coli Ten millilitres of Luria–Bertani medium with 50 lgÆmL)1 kanamycin was inoculated (1%) from overnight cultures
of E coli BL21(DE3) containing either pWUR66, pWUR67 or pWUR68 After growth at 37C to
D600¼ 0.8, 0.5 mM isopropyl thio-b-D-galactoside was added to induce expression After overnight growth, 2-mL
of each culture was centrifuged (10 min at 20 000 g) and cells were resuspended in 50 mMTris/HCl buffer pH 7.8 Cells were sonicated and the supernatants were subse-quently heated for 20 min at 70C to denature E coli proteins The supernatants were analysed by SDS/PAGE and by activity measurements
For enzyme purification 2-L cultures were grown in essentially the same way as described above Cells (5–6 g wet weight) were harvested by centrifugation (2200 g for
15 min at 10C) and resuspended in 28 mL 50 mM Tris/ HCl buffer pH 7.8 The suspension was passed twice
Fig 1 Phylogenetic tree of NADH oxidases and related enzymes The
tree was constructed from alignments using the CLUSTAL method [20]
of the Megalign program ( DNASTAR , London, UK) and Nox sequences
available at the NCBI data base The units at the bottom indicate the
number of substitution events Genbank indentifiers are indicated in
parentheses.
Trang 3through a French press (110 MPa) and the resulting crude
cell extract was used for purification of the recombinant
NADH oxidases
Purification of recombinant NoxA-1 and NoxB-1
The E coli cell extract was heated for 30 min at 70C
(NoxA-1) or for 30 min at 50C (NoxB-1), and denatured
proteins were pelleted by centrifugation (17 200 g for
15 min at 10C) This pellet fraction was washed with
10 mL Tris/HCl buffer pH 7.8 and the centrifugation step
was repeated The supernatants of both centrifugation steps
were combined, filtered through a 0.45-lm filter and loaded
onto a Q-Sepharose column (1.6· 10 cm) equilibrated with
20 mMTris/HCl buffer pH 7.8 Bound proteins were eluted
by a 200-mL linear gradient of NaCl (0–1M in Tris/HCl
buffer pH 7.8) NoxA-1 eluted in a single peak at 0.38M
NaCl In a similar purification NoxB-1 eluted at 0.15M
NaCl Active fractions were pooled and applied to a
hydroxyapatite column (Bio-Gel HT; 1.6· 10 cm)
equili-brated with 10 mM sodium phosphate buffer pH 7.0
Elution was performed with a 200-mL gradient from 10
to 500 mMsodium phosphate (pH 7.2) The NoxA-1 as well
as the NoxB-1 eluted right after the flow-through fraction
Active fractions were pooled and concentrated by
ultrafil-tration (Filtron; 10 kDa cut-off) A 200-lL aliquot of the
concentrated samples was loaded onto a Superdex-200 HR
column, equilibrated in 20 mMTris/HCl pH 7.8 containing
150 mM NaCl NoxA- 1 as well as NoxB- 1 eluted as two
overlapping activity peaks
PAGE
The purity of the various purification fractions was regularly
checked by SDS/PAGE according to the procedure of
Laemmli using 15% (w/v) gels [16] Protein samples were
denatured by heating in SDS-sample buffer for 5 min at
100C SDS/PAGE was also used to determine the subunit
molecular mass Calibration was performed using a set of
calibration proteins: myosin (200 kDa), b-galactosidase
(116.25 kDa), phosphorylase b (97.4 kDa), serum albumin
(66.2 kDa), ovalbumin (45 kDa) and carbonic anhydrase
(31 kDa) Protein bands were stained with Coomassie
brilliant blue R250
Enzyme assays
NADH oxidase activity was measured
spectrophoto-metrically in 1-mL quartz cuvettes on a Hitachi U-2010
spectrophotometer equipped with a thermostatted cuvette
holder Initially, one standard method was used for
measuring NADH oxidase activity of the recombinant gene
products The standard assay mixture contained 100 mM
potassium phosphate buffer (pH 7.0), 0.06 mM FAD,
0.29 mM NADH and an appropriate amount of enzyme
The activity was determined by monitoring the oxidation of
NADH at 334 nm and at 70C (e334¼ 6.18 mM )1Æcm)1)
[17] In addition, separate assays were used for determining
NoxA-1 and NoxB-1 activity, which were performed at
80C and 70 C, respectively NoxB-1 was less stable than
NoxA-1, and therefore the assays were performed at 70C
instead of 80C
For NoxA-1 the assay mixture contained potassium phosphate/sodium citrate buffer (50 mM each; pH 8.0), 0.06 mM FAD, 0.29 mM NADH and an appropriate amount of enzyme For NoxB-1 the same assay mixture was used, except that the pH was adjusted to 6.5 Specific activities were calculated from the initial linear change in absorbance Absorbance changes were corrected for non-enzymatic NADH conversion Where indicated FAD was omitted from the assay mixture
When electron acceptors other than oxygen were tested, all assay constituents were made anoxic by repeated evacuation and gassing with N2 gas in stoppered serum bottles The stoppered cuvettes were also evacuated and gassed with N2, and the different components were added
by syringe The following extinction coefficients were used
to calculate the specific activities: ferricyanide, 1.00 mM )1at
420 nm; 2,6 dichlorophenolindophenol (DCPIP), 20 mM )1
at 600 nm; 5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB), 13.6 mM )1 at 412 nm, benzyl viologen, 8.6 mM )1 at
578 nm; cytochrome c, 21.1 mM )1at 550 nm For menadi-one and 2,3-dimethyl-1,4-naphthoquinmenadi-one extinction coef-ficients were determined as 2.72 and 2.39 mM )1at 334 nm, respectively, based on a 1 : 1 stoichiometry with NADH
To investigate whether the NADH oxidases produced either H2O or H2O2, an activity assay without FAD was run
to completion at 50C and a 50-lL sample from the reaction mixture was tested in a separate assay at 22C for the presence of H2O2 This second mixture (1 mL) con-tained potassium phosphate/sodium citrate buffer (50 mM
each, pH 6.5), 0.5 mM 3,3¢-dimethoxybenzidine, and 7 U horseradish peroxidase The increase in absorbance (460 nm) was compared to a standard curve, which was prepared separately using known amounts of H2O2 The assay was not disturbed by residual NADH, which might have remained in the Nox assay The decrease in NADH
in the first assay was related to the amount of H2O2found
in the second assay
Protein was determined according to Bradford [18] using the Bio-Rad protein assay kit, with BSA as standard Analysis of catalytic properties
Kinetic parameters of NoxA-1 and NoxB-1 were deter-mined in the specific assay systems, by measuring the initial rate at different starting concentrations of NADH in the presence of ambient dissolved oxygen concentrations Km (for NADH) and Vmax values were obtained by a computer-aided direct fit to the Michaelis–Menten curve (TABLE CURVE 2D) The Kmvalues for O2 were determined from one single assay, in which the stoppered cuvettes were completely filled with the specific assay buffers The buffers, which were equilibrated at 60C were calculated to contain 0.135 mM of dissolved oxygen [19] The reaction was started upon addition of anoxic NADH The decrease in oxygen concentration was calculated from the decrease in NADH, assuming that for each mole of NADH one mole
of O2was required (in case only H2O2was produced) The reaction rates at the various O2 concentrations were subsequently fitted using the Michaelis–Menten equation
It was assumed that the NADH concentration was saturating, meaning that only apparent Km values were obtained
Trang 4The temperature dependence of NoxA-1 and NoxB-1
was determined in the range 20–90C The pH-dependence
was determined in the standard potassium phosphate/
sodium citrate buffer at 80C or 70 C, for NoxA-1 or
NoxB-1, respectively
Stability analysis
The thermostability of the enzymes was tested by incubating
the purified enzyme in potassium phosphate buffer
(100 mM, pH 7.0) in a closed vial in a water bath at
80C At regular time intervals a sample was taken and
tested in the standard assay For NoxB-1 the stability was
determined also in the presence of 2 mM dithiothreitol
Half-life values were calculated from a fit of the data
(exponential decay: y¼ aÆe–bx)
Results
Characterization based on amino acid sequence
The sequences of the three NADH oxidases from A
fulgi-dusthat were investigated here were aligned with various
other NADH oxidase sequences, available at the NCBI
database The resulting phylogenetic tree (Fig 1) clearly
showed that the three NADH oxidases belong to different
phylogenetic clusters NoxA-1 falls within a group of typical
NADH oxidases of 49 kDa, including the well-studied
NADH oxidases from Enterococcus feacalis and
Strepto-coccus mutans[3,21] This group also contains a NADH
peroxidase, which performs a NADH-dependent reduction
of H2O2 to H2O, and a coenzyme A disulfide reductase,
which catalyses the NADPH-dependent reduction of
CoA-S-S-CoA to CoA-SH [4,5] The NADH oxidase and
-peroxidase are H2O-forming enzymes and are believed to
play a role in oxidative stress defence or in the recycling of
oxidized pyridine nucleotides The CoA disulfide reductase
is involved in maintaining a sufficiently high intracellular
thiol concentration [5] The alignment (not shown) revealed
conserved binding motives for FAD or NAD(P) and,
moreover, most sequences in this group contain a redox
active cysteine, which is regarded essential for the reduction
of O2 to H2O and for the disulfide reduction [22] The
NoxA-4 from A fulgidus (GI:11498556), a NADH oxidase
gene from P horikoshii (GI:14590747), and three genes
from Sulfolobus solfataricus (GI:15899854, GI:15897884,
GI:15899669) do not contain this cysteine
NoxB-1 belongs to a small group of 72-kDa proteins, of
which several are involved in the reduction of unsaturated
acids or aldehydes For instance, enoate reductase (enr)
from Clostridium tyrobutyricum, 2,4 dienoyl-CoA reductase
(fadH) from E coli, and NADH:flavin oxidoreductase
(baiH) from Eubacterium sp strain VPI 12708, all perform a
NAD(P)H-dependent reduction of a carbon–carbon double
bond [23–25] This group also contains the NADH oxidase
of T brockii, but no physiological role has been ascribed to
it [7] The alignment revealed potential ligands for an iron–
sulfur cluster and FAD or NAD(P) binding domains
(Fig 2)
NoxC belongs to a group of small 20-kDa proteins, that
form H2O2 instead of H2O The NADH oxidases from
Thermus aquaticusand Thermus thermophilus also belong to
this group [7,8] The physiological role of these enzymes is not known
Cloning and expression All three nox genes gave gene products when expressed in
E coli BL21 (DE3) as judged by SDS/PAGE (Fig 3) NoxA-1 gave a clear band of the expected size (48 kDa) Expression of noxB-1 was less clear, but still visible on the gel The expected molecular mass based on the sequence of NoxB-1 is 68 kDa NoxC appeared as two proteins of
36 kDa and 20 kDa The larger protein may represent undenatured NoxC, because upon prolonged boiling in SDS-sample buffer, it disappeared and the expected 20-kDa protein increased (data not shown) Cell-free extracts of
E coliproducing NoxA-1 and NoxB-1 showed significant NADH oxidase activity (see below) For NoxC, however,
no NADH oxidase activity was measured Also, in the presence of FMN instead of FAD, or NADPH instead of NADH no NoxC activity was found For this reason, further purification and characterization of NoxC was abandoned
Purification of the recombinant enzymes NoxA-1 and NoxB-1 were purified essentially by the same procedure The first step that capitalized on their thermo-stability was a heat treatment, resulting in the removal of
E coliproteins Whereas this treatment worked fine with the NoxA-1, NoxB-1 remained contaminated with E coli proteins Four subsequent chromatographic steps were necessary to obtain a homogeneous preparation (Tables 1 and 2) Gelfiltration of NoxA-1 on Superdex 200 resulted in two activity peaks, corresponding to molecular masses of the native NoxA-1 of approximately 94 and 178 kDa, which suggests that the NoxA-1 exists as dimer and to some extent as tetramer Most homologous NADH oxidases from mesophiles (Enterobacter feacalis, Streptococcus mutans and Serpulina hyodysenteria) are monomers or dimers [3,21,26] A NADH oxidase from P furiosus, which was recently described, also existed as dimer [10] Gelfiltra-tion of NoxB-1 resulted in a major activity peak with a shoulder, corresponding to molecular masses of approxi-mately 70 and 152 kDa These data suggested that the NoxB-1 exists as monomer and for a minor part as dimer Other homologous enzymes have been shown to have different quaternary structures, like a trimer (Eubacterium sp.), hexamer (T brockii) or dodecamer (Clostridium tyrobutyricum) [6,23,25]
Catalytic properties The NADH oxidase activity of NoxA-1 and NoxB-1 was stimulated upon addition of FAD (60 lM) to the assay mixture For NoxA-1 this stimulation was 2.5 fold, for NoxB-1 3.7-fold Addition of FMN instead of FAD did not stimulate NADH oxidase activity This result suggested that both Nox enzymes contain FAD as prosthetic group, and that part of the protein had apparently lost its cofactor Indeed, during the purification of especially NoxA-1, it was observed that the yellow colour of the enzyme fractions gradually disappeared From the UV/visible spectrum of
Trang 5NoxA-1 (and NoxB-1), with an absorbance maximum at
450 nm, it could be concluded that a flavin is present (data
not shown)
For NoxA-1 and NoxB-1 different pH optima of 8.0 and
6.5 were found, respectively (Fig 4) At their pH optimum,
apparent Vmaxvalues were found of 8.7 ± 0.5 UÆmg)1for
NoxA-1 and 1.5 ± 0.03 UÆmg)1 for NoxB-1 The latter
activity was found to be strongly influenced by the
presence of mercaptans Dithiothreitol (DTT; 2 mM) or
b-mercaptoethanol stimulated NoxB-1 activity up to
two-fold In contrast, NoxA-1 activity was inhibited by DTT
(2 mM), e.g in its presence the activity rapidly decreases to
<10% of the activity without DTT The affinity for NADH
was the highest for NoxB-1 (apparent Km¼ 0.011 ± 0.001 mM) (Fig 5) NoxA-1 showed a rather low affinity for NADH (apparent Km¼ 0.13 ± 0.014 mM) compared
to NoxB-1 and to other thermoactive NoxA homologues from P furiosus (Km< 4 lM) or A fulgidus (NoxA-2:
Km¼ 3.1 lM) NoxA-1 and NoxB-1 did not show activity with NADPH
For NoxA-1 an apparent Kmfor oxygen of 0.06 ± 0.03 was determined The Km value for oxygen of NoxB-1 appeared to be much higher and for this reason difficult to assess The fit-program resulted in an apparent Km of 2.9 mM, far above the maximum dissolved oxygen concen-tration (0.086 mMat a pO of 0.2 105Pa at 80C)
Fig 2 Multiple alignment of NoxB-1 homologues Conserved and moderately conserved residues are shaded black or grey Putative NAD-or FAD-binding motifs are boxed Cysteine residues of a putative ferredoxin-like motif are indicated by arrowheads The abbreviations used are as follows (Genebank identifier in parentheses): Nox Tbro, NADH oxidase of Thermoanaerobacter brockii (GI:48123); DienoylCoA, 2,4-dienoyl-CoA reductase of E coli (GI:1176118); NADH:flav, NADH: flavin oxidoreductase of Eubacterium sp (GI:416702); Enoate red, enoate reductase of Clostridium acetobutylicum (GI:15026455); Nox Sso, NADH oxidase (SSO2025) of Sulfolobus solfataricus (GI:15898816).
Trang 6Addition of EDTA to the assay mixture did not cause a
decrease in activity of NoxA-1 or NoxB-1, suggesting that
divalent cations are not required for activity
Effect of temperature on activity
For NoxA-1and NoxB-1 an identical temperature optimum
of 80C was found, corresponding to the physiological
growth optimum of the organism (Fig 6) Up to 70C, the
increase in NADH oxidase activity followed a linear
Arrhenius plot (ln k vs 1/T; data not shown), from which
activation energies of 76.6 kJÆmol)1 and 52.2 kJÆmol)1
could be calculated for NoxA-1 and NoxB-1, respectively
Despite the identical temperature optima, the thermostabi-lity of both Nox enzymes at this temperature was consid-erably different Whereas NoxA-1 showed a half-life of
40 h at 80 C (data not shown), NoxB-1 showed a half-life of only 40 min For NoxB-1, the temperature stability was investigated in the presence and absence of DTT In addition to the stimulating effect on the absolute activity, DTT also raised the stability about twofold (half-life
83 min) (Fig 7)
The product of the NADH oxidase reaction NADH oxidases can perform the bivalent reduction of oxygen to H2O2or the tetravalent reduction of oxygen to
H2O Production of H2O2was tested by analysing the assay mixture in a separate peroxidase assay, using horseradish peroxidase and 3,3¢-dimethoxybenzidine as electron donor
In the NoxA-1 assay between 71% and 95% of the amount
Fig 3 SDS/PAGE of extracts of recombinant E coli containing
NoxA-1, NoxB-1 or NoxC from A fulgidus M, Calibration proteins.
The molecular mass of the calibration proteins is indicated (kDa).
Table 1 Purification scheme of NoxA-1 from A fulgidus Activities were determined in phosphate/citrate buffer pH 8.0.
Total volume (mL)
Protein (mgÆmL)1)
Total protein (mg)
Specific activity (UÆmg)1)
Total activity (U)
Purification (fold)
Recovery (%) Crude extract 28 21.5 602 0.59 355.2 1.0 100 Heat-treatment 30.4 3.55 108 3.29 355 5.57 100 Q-sepharose 47.54 1.32 62.75 4.69 294 7.94 82.7 Hydroxyapatite 44.9 1.06 47.61 4.69 223.3 7.94 62.8 Macrosep 6.92 6.62 45.79 5.07 232 8.59 65.3 Superdex 200 93.38 0.298 27.83 5.82 161.9 9.86 45.6
Table 2 Purification scheme of the NoxB-1 from A fulgidus Activities were determined in phosphate/citrate buffer (pH 6.5).
Total volume (mL)
Protein (mgÆmL)1)
Total protein (mg)
Specific activity (UÆmg)1)
Total activity (U)
Purification (fold)
Recovery (%) Crude extract 28 20.84 583.5 1.139 664.6 1.0 100 Heat-treatment 28.5 6.16 175.56 2.35 412.56 2.06 62.1 Q-sepharose 60.16 2.09 125.7 2.52 316.8 2.21 47.7 Hydroxyapatite 83.8 0.67 56.13 3.43 192.5 3.01 29 Macrosep 7.74 8.2 63.5 2.27 144.16 1.99 21.7 Superdex 200 196.9 0.153 30.1 4.05 122 3.55 18.3
Fig 4 pH dependence of purified NoxA-1 (s) and NoxB-1 (d).
Trang 7of NADH which was converted was recovered as H2O2 In
the NoxB-1 assay, this value amounted to 97% FAD was
omitted from these assays, because unbound FAD may
facilitate H2O2 production via nonenzymatic oxidation of FADH2 [27] These results indicate that both enzymes probably produce exclusively H2O2 The fact that the recovery of H2O2was sometimes less than 100% (70–80%), could be explained by the observation that the amount of
H2O2measured, was influenced by the time period between the NADH conversion and the actual measurement of
H2O2 Apparently, the amount of H2O2in the assay mixture slowly decreased, despite the absence of NADH, which was already completely converted at that moment
Electron acceptors other than oxygen Because the physiological role of NoxA-1 and NoxB-1
is not known, it was of interest to test various electron acceptors other than oxygen The results of these assays, which were performed in the absence of oxygen, are summarized in Table 3 Flavines, like FAD, are known to
be able to react with various one-or two-electron acceptors
In accordance, NoxA-1 and NoxB-1 show activity with several of the e-acceptors tested DCPIP, ferricyanide, menadione and 2,3-dimethyl-1,4-naphthoquinone are e-acceptors commonly used in the detection of membrane bound dehydrogenases However, the activities were rather low (see Discussion) NoxA-1 also showed some activity with a cytochrome c, but again the activity is low One of the homologues of NoxA-1 has been identified as NADH peroxidase [4] For this reason H2O2was tested as electron acceptor under anoxic conditions Neither NoxA-1 nor NoxB-1 showed convincing NADH peroxidase activity Another NoxA-1 homologue has recently been recog-nized as CoA disulfide reductase, an enzyme that performs a
Fig 5 Rate dependence of NoxA-1 and NoxB-1 on the NADH
con-centration Data points were fitted according to the Michaelis–Menten
equation.
Fig 6 Temperature dependence of purified NoxA-1 (s) and NoxB-1
(d).
Fig 7 Thermal stability of NoxB-1 The purified enzyme was
incu-bated at 80 C in 100 m M phosphate buffer pH 7.0 in the presence (d)
and absence (s) of 2 m M DTT.
Table 3 Specific activity of NoxA-1 and NoxB-1 with different electron acceptors.
Presence
of FADa
NoxA-1 (UÆmg)1)
NoxB-1 (UÆmg)1)
Ferricyanide – 5.8 1.8 Menadione – 1.29 1.93 2,3-Dimethyl-1,4-naphthoquinone – 1.56 1.29 Cytochrome c – 0.03 0 Benzyl viologen + 4.5 1.3
Coenzyme A (oxidized) +/– 0 0 Glutathione (oxidized) +/– 0 0
Polysulfide + 0 0.65b Tiglic acid +/– NT 0 Cinnamic acid +/– NT 0 Crotonate +/– NT 0
a Activity was determined in the presence (+) or absence (–) of FAD +/–, Substrates were tested with and without FAD.bOnly
in 100 m M Tris/CL buffer pH 7.8 at 60 C NT, not tested; tiglic acid, trans-2-methyl-2-butenoic acid; cinnamic acid, 3-phenyl-2-propenoic acid; crotonate, 2-butenoate; menadione, 2-methyl-1,4-naphthoquinone.
Trang 8disulfide reductase activity via a single cysteine residue [5].
For this reason, NoxA- 1 as well as NoxB- 1 were tested
using DTNB as e-acceptor An activity of 3.68 UÆmg)1was
determined for NoxA-1 NoxB-1 also showed a significant
disulfide reductase activity of 0.52 UÆmg)1 Both activities
were determined in the presence of FAD In the absence of
FAD, the reduction of DTNB was substantially less Other
disulfides of more physiological nature like oxidized
Coen-zyme A, glutathione or cystine did not cause a NADH
oxidation in the absence of oxygen, nor did they stimulate
the DTNB reduction
A special type of electron acceptor tested here was
polysulfide Polysulfide is a soluble form of elemental sulfur,
which has been shown to act as electron acceptor by various
hyperthermophiles NoxB-1, but not NoxA-1, showed a
significant NADH-dependent polysulfide reductase activity
of 0.65 UÆmg)1 However, this activity is again low when
compared to other polysulfide reductases, like the
NADPH-dependent sulfide dehydrogenase (7.0 UÆmg)1) from
P furiosus[28]
Because NoxB-1 showed homology to enzymes involved
in the reduction of unsaturated acids or aldehydes (Fig 2),
a few model substrates were tested as potential electron
acceptors (Table 3) However, none of these caused an
oxidation of NADH when added to the anoxic reaction
mixture
Discussion
The noxA-1 and the noxB-1 gene from the
hyperthermo-philic archaeon A fulgidus were successfully cloned and
expressed in E coli The alignment to homologous genes in
the database revealed that NoxA-1 and NoxB-1 belong to
different phylogenetic groups (Fig 1) Nevertheless, the
similarity to other NADH oxidase gene sequences does not
simply lead to their physiological function For instance,
NoxA-1 belongs to the family of pyridine nucleotide
disulfide oxidoreductases (Pfam), which contains enzymes
that may function as NADH oxidase, NADH peroxidase
or as CoA disulfide reductase The various electron
acceptors tested here did not indicate an obvious function
(Table 3) The reduction of DCPIP and ferricyanide
suggests that NoxA-1 may have a role as NADH
dehydrogenase as part of the electron transport chain for
sulfate reduction Moreover, it has recently been suggested
that NoxA-2 of A fulgidus may also function in electron
transport for sulfate reduction, because the enzyme
copu-rified with D-lactate dehydrogenase and both enzymes
colocalized to the periplasmic side of the membrane [9,30]
However, the activities found here for NoxA-1 towards
menadione, 2,3-dimethyl-1,4-naphthoquinone and
cyto-chrome c are rather low, and thus do not support this
hypothesis For example, the F420H2: quinone
oxido-reductase from A fulgidus showed specific activities of
96 UÆmg)1and 92 UÆmg)1with
2,3-dimethyl-1,4-naphtho-quinone and menadione, respectively [29] A novel type
menaquinone, present in the membrane fraction of
A fulgidus, probably acts as the physiological electron
acceptor [31]
NoxA-1 showed substantial disulfidereductase activity
(3.7 UÆmg)1), but this DNTB-reducing activity was not
stimulated by disulfides like oxidized coenzyme A,
gluta-thione or cystine The activity was, however, strongly stimulated upon addition of FAD, which indicated that free FAD was involved in the reduction of DNTB This suggests that the observed disulfide reduction may not be the physiological role of NoxA-1 Moreover, when we compare the disulfide reductase activity of NoxA-1 with that of a true disulfide reductase (CoA disulfide reductase from Staphylo-coccus aureus; Spec activity¼ 4570 UÆmg)1), the latter is at least 1000-fold more active [5] In this respect we also tested whether NoxA-1 exhibited polysulfide reductase activity Polysulfide is a soluble form of sulfur consisting of predominantly tetrasulfide (S42–) and pentasulfide (S52–), and it is assumed that an S–S bond is cleaved similar to the disulfide reduction This activity was of special interest because a similar NADH oxidase (NoxA-2) of P furiosus was shown by DNA microarray analysis to be strongly up-regulated (7.4-fold) when cells were grown in the presence of sulfur [32] The expression of two other ORFs
in the P furiosus genome increased more than 25-fold, and their products termed SipA and SipB are proposed to be part of an S-reducing protein complex Although A fulgidus
is not able to grow by sulfur reduction, its genome contains homologues of the SipA and SipB encoding genes Unfor-tunately, NoxA-1 did not show polysulfide reductase activity Nevertheless, the similarity to the S-upregulated NoxA-2 of P furiosus and to the membrane associated NoxA-2 of A fulgidus, suggests some respiratory role Alternatively, the function of NoxA-1 may actually be that of an NADH oxidase, using the reducing power of NADH to remove traces of oxygen that otherwise may lead to harmful oxygen species like O2, H2O2, or OHÆ The Kmfor oxygen of NoxA-1 is 60 lM, which is not very low compared to the amount of oxygen that can maximally dissolve at 80C (102 lM at ambient oxygen concentrations and average marine salinity) However, because A fulgidus is a strict anaerobe, it will most likely have to deal with much lower oxygen concentrations A role as detoxicant has also been proposed for the NADH oxidase (NOX1) from the hyperthermophile P furiosus [10] The affinity for oxygen of the latter enzyme, however, is even lower (a Km of at least 110 lM has been reported), which makes this enzyme also not very efficient if it is assumed to remove small amounts of oxygen in an anaerobic environment Unfortunately, oxygen affinity data of true NADH oxidases are not available in the literature, making a comparison impos-sible The most plausible argument against a role as detoxicant, however, is the fact that the NoxA-1 and also the enzyme from P furiosus produce predominantly
H2O2 Thus, instead of preventing oxidative stress through
O2removal, these Nox enzymes aggravate the problem by producing H2O2 On the other hand, H2O2 which is produced by the Nox, may be converted further by a catalase-peroxidase, which has also been demonstrated in
A fulgidus[33] But in this case H2O2is converted back to
O2, which combined with the NADH oxidase lowers the amount of oxygen by only 50% The Km for oxygen of NoxB-1 is even much higher ( 3 mM), making a role as oxygen detoxifying system very unlikely Moreover, also NoxB-1 produces H2O2 instead of H2O
In a recent paper Abreu et al describe a superoxide scavenging system in A fulgidus, and propose that
Trang 9NAD(P)H oxidases may have a role in oxygen
detoxifica-tion, not by directly reducing oxygen, but via intermediate
redox enzymes like rubredoxin and neelaredoxin [34] A
similar system has been proposed for Desulfovibrio gigas
[35] This hypothesis certainly deserves further attention, but
requires purification of the rubredoxin and neelaredoxin
The production of H2O2is in contrast with the data on
mesophilic homologues of NoxA-1 or NoxB-1, which all
produce H2O Also the NoxA-2 from A fulgidus, the
NADH oxidase from P furiosus, and the NADH oxidase
from T brockii have been demonstrated to form H2O2
instead of the usual H2O [9,10] Thus, possibly the
production of H2O2is a thermophilic feature It has been
put forward that reduction of oxygen to H2O2may be an
artefact, because in anaerobes the flavin moiety of
flavo-proteins is exposed to the solvent and can easily transfer
electrons to oxygen to form H2O2 In aerobes the flavin is
protected from this unwanted oxygen reduction, because
the flavin is buried in the protein Remarkably, for the
NADH oxidase from P furiosus only 61% of the NADH
was recovered as H2O2 (NADH/H2O2 ratio of 0.61),
suggesting that the enzyme produced both H2O2and H2O
[10] Occasionally, we also found <100% recovery of H2O2
compared to NADH, but this could be diminished by
shortening the time period between the Nox assay and the
H2O2-assay Possibly, this also applies for the enzyme from
Pyrococcus
Concerning the physiological role of the NoxA-1, the
direct neighbourhood of noxA-1 in the genome was
investigated A CTP synthase, a GMP synthase and several
hypothetical proteins accompany noxA-1, which do not
provide insight as to the physiological role of the NoxA-1 A
STRING analysis [36] of the surrounding genes, however,
reveals that all NoxA and NoxB homologues have a
predicted redox protein, regulator of disulfide bond
forma-tion (COG0425) in their neighbourhood This COG belongs
to a functional category involved in post-translational
modification, protein turnover, and chaperones, which also
does not reveal a physiological role of the Nox enzymes
Recent experiments by Pagala et al [30] have shown that
whole cell extracts of A fulgidus exhibit multiple NADH
oxidase activities, as judged by renatured SDS/PAGE gels
It was concluded that the majority of the Nox enzymes
in A fulgidus are expressed constitutively under strictly
anaerobic conditions The fact that the expression of the
Nox enzymes is not regulated, also suggests that they have
some fundamental metabolic role, and not an occasional
role during oxygen stress
NoxB-1 shows homology to a small group of enzymes
that is involved in the reduction of unsaturated acids or
aldehydes For instance, enoate reductase (enr) from
Clostridium tyrobutyricum, 2,4 dienoyl-CoA reductase
(fadH) from E coli, and NADH:flavin oxidoreductase
(baiH) from Eubacterium sp strain VPI 12708, all perform a
NAD(P)-dependent reduction of a carbon–carbon double
bond [23–25] However, several commonly used
unsatur-ated compounds, like tiglic acid, cinnamic acid or crotonate
did not show any activity when tested with NoxB-1
Possibly, the enzyme requires CoA-activated unsaturated
compounds, which have not been tested here As mentioned
above, the adjacent genes of NoxB-1 do not reveal any
information concerning its function On the other hand, the
gene encoding NoxB-2 of A fulgidus, which is 98.9% identical to NoxB-1, lies upstream of a gene encoding a medium-chain acyl-CoA ligase, suggesting a role in fatty acid and phospholipid metabolism
Thus, despite an extensive analysis of the catalytic capabilities of NoxA-1 and NoxB-1, no obvious physio-logical role can be ascribed to them Further studies, for instance using Northern blots or DNA microarrays may indicate conditions at which the enzymes are expressed and thereby unveil their cellular function
Because both NADH oxidases produce H2O2instead of
H2O, they may find application in biosensors as mediator between a dehydrogenase and the electrode For this purpose the enzymes should have sufficient stability and appropriate kinetics Although, the stability of NoxB-1 is considerably lower than that of NoxA-1 (stable at 80C for
1 h and 40 h, respectively), the stability is likely to be sufficient at more moderate temperatures Concerning the catalytic activity, kcat/Kmvalues of 0.053· 106
M )1Æs)1and 0.156· 106
M )1Æs)1 can be calculated for NoxA-1 and NoxB-1, respectively These catalytic efficiencies are sub-stantially lower than the value found for the NADH oxidase
of Thermus thermophilus, which was determined at room temperature (kcat/Km¼ 1.250 · 106M )1Æs)1) [37] Thus, compared to the latter enzyme, which also forms H2O2 and which is also reasonably stable, NoxA-1 and NoxB-1 are less suited for biosensor application
Acknowledgements
This work was partly funded by the European Community under the Industrial & Materials Technologies Programme (Brite-Euram III) (Contract BRPR-CT97-0484).
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