A novel NADPH-dependent oxidoreductase with a unique domain structure in the hyperthermophilic Archaeon, Thermococcus litoralis

R E S E A R C H L E T T E R

AnovelNADPH-dependentoxidoreductasewithaunique domainstructure in the hyperthermophilicArchaeon,ThermococcuslitoralisAndras Toth1, Maria Takacs2, Geza Groma1, Gabor Rakhely1,2 & Kornel L. Kovacs1,2

1Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary; and 2Department of Biotechnology, University of

Szeged, Szeged, Hungary

Correspondence: Kornel L. Kovacs,

Department of Biotechnology, University of

Szeged, H-6726, Kozep fasor 52, Szeged,

Hungary. Tel.: 136 62 544 351; fax: 136 62

544 352; e-mail: [email protected]

Received 29 August 2007; accepted 3 January

2008.

First published online 18 March 2008.

DOI:10.1111/j.1574-6968.2008.01085.x

Editor: Christiane Dahl

Keywords

hyperthermophilic Archaea; Thermococcus

litoralis ; sulfur oxidoreductase.

Abstract

Thermococcus litoralis, a hyperthermophilic Archaeon, is able to reduce elemental

sulfur during fermentative growth. An unusual gene cluster (nsoABCD) was

identified in this organism. In silico analysis suggested that three of the genes

(nsoABC) probably originated from Eubacteria and one gene (nsoD) from Archaea.

The putative NsoA and NsoB are similar to NuoE- and NuoF-type electron

transfer proteins, respectively. NsoC has a unique domain structure and contains a

GltD domain, characteristic of glutamate synthase small subunits, which seems to

be integrated into a NuoG-type sequence. Flavin and NAD(P)H binding sites and

conserved cysteines forming iron–sulfur clusters binding motifs were identified in

the protein sequences deduced. The purified recombinant NsoC contains one FAD

cofactor per protein molecule and catalyzes the reduction of polysulfide with

NADPH as an electron donor and it also reduces oxygen. It was concluded that the

Nso complex is a new type of NADPH-oxidizing enzyme using sulfur and/or

oxygen as an electron acceptor.

Introduction

Thermococcus litoralis, growing optimally at 85 1C, is a

member of the archaeal Thermococcales order (Neuner

et al., 1990). These are heterotrophic hyperthermophiles

utilizing carbohydrates and peptides and produce acetate,

CO2 and H2 or, in the presence of sulfur, H2S, as major end

products (Fiala & Stetter, 1986; Neuner et al., 1990). During

fermentative metabolism, extensively studied in Pyrococcus

furiosus and also in T. litoralis, reduced ferredoxin and

NADPH are generated (Adams & Kletzin, 1996). To main-

tain the catabolic pathways, oxidized ferredoxin and

NADP1 pools must be replenished. A possible way of

disposing of excess reductants is the transfer of electrons to

the terminal electron acceptor S0 catalyzed by sulfur reduc-

tase enzymes.

At present, the sulfur metabolism of P. furiosus is the best

known among Thermococcales species. It has four character-

ized enzymes that possess sulfur reductase activity in vitro:

two heterotetrameric [NiFe] hydrogenases (Hyh1, Hyh2),

which are also named sulfhydrogenases (Ma et al., 1993,

2000), the heterodimeric sulfide dehydrogenase (Sud) (Ma

& Adams, 1994) and the monomeric coenzyme A-depen-

dent NAD(P)H sulfur oxidoreductase (NSR) (Schut et al.,

2007). While the involvement of NSR in S0 reduction in vivo

has been suggested (Schut et al., 2007), neither the hydro-

genases nor Sud might play a role in S0 reduction within the

cells (Schut et al., 2001).

In T. litoralis, the molecular and biochemical background

of sulfur metabolism is poorly understood. Thus far, only a

soluble [NiFe] hydrogenase (Hyh1) has been characterized

in this archaeon, which has sulfur reductase activity

(Rakhely et al., 1999). In this paper, we report on the

molecular and biochemical characterization of a new, un-

ique sulfur reductase in T. litoralis.

Materials and methods

Strains and growth conditions

Thermococcus litoralis DSM5473 were grown under anaero-

bic conditions at 85 1C in a complex medium, as described

previously (Diruggiero et al., 2000), in the presence (0.1%,

w/v) or in the absence of sulfur.

FEMS Microbiol Lett 282 (2008) 8–14c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Escherichia coli XL1-BlueMRF’ and BL21(DE3)Codon-

Plus-RIL (Stratagene) strains were cultivated in Luria–

Bertani medium (LB) at 37 1C (Ausubel et al., 1996).

Antibiotics were used at the following concentrations:

ampicillin (100mg mL�1) and streptomycin (25 mg mL�1).

DNA techniques

DNA manipulation steps were carried out using general

procedures (Ausubel et al., 1996) or the manufacturers’

instructions. Site-specific mutagenesis was performed by

the QuikChange II XL Site-Directed Mutagenesis Kit

(Stratagene).

Isolation, cloning and sequencing of the nsooperon

The genomic region of T. litoralis upstream from the hyh2

operon (for accession number see: Fig. 1) was cloned and

sequenced by chromosomal walking (Fig. 1a). Partial geno-

mic libraries created from XbaI and SphI digested genomic

DNA in pBluescript SK1 were screened for overlapping

clones, using digoxigenin-labelled PCR probes obtained

with LSHO10/LSHO11 (LSHO10: 50-AAGGACGTTCCTG

TCCATGAAG-30; LSHO11: 50-AGTCGGCATGAACTCTT

GTGGA-30) and LSHO14/LSHO18 (LSHO14: 50-TCCTCT

GCTTCCTCAACTTC-30; LSHO18: 50-AATAGAGGCGAT

GCCAGAAC-30) primer pairs. These 3926-bp (pLU9) and

5103-bp (pLS91) genomic fragments were subcloned and

sequenced on both strands by primer walking.

RNA isolation, reverse transcriptase (RT)-PCR andprimer extension

Total RNA extracted from T. litoralis using TriReagent

(Sigma) was treated with RNAse-free DNAse I (Fermentas).

In RT-PCR experiments, MMLV RT (Promega) was used

to synthesize cDNA from 1mg of total RNA using the primer

LSHO37 (50-TAATCCTCTGCACTCCTCTC-30) matching

to the 30 end of the nsoD transcript. Then, the PCR was

performed with the following primers (amplifying part of

the nsoA gene): LSHO1 (50-CGCAGCAATTCCACAAGA

GT-30) and LSHO2 (50-AGAATGGTGGGTGGTAATCA-30).

The transcriptional start point of the nso operon was

determined by primer-extension experiments with the

LSHO51 (50-AGGCAGTTTTAGGTATTTTGAAATCTCC

TC-30) end-labelled primer (Ausubel et al., 1996).

Expression and purification of rNsoC

For construction of the vector expressing the NsoC protein

fused to the tandem FLAG-tag-Strep-tag II at the C terminus

(designated as rNsoC), nsoC was amplified from genomic

DNA in two parts with the LSHO38/LSHO39 and LSHO40/

LSHO41 primer pairs (LSHO38: 50-CATATGGTCAAGCT

CATCGTGAAT-30; LSHO39: 50-GTAATAGGCGCATG

CAAGT-30; LSHO40: 50-GACTTGCATGCGCCTATTAC-30;

LSHO41: 50-GAGGGAGAATATCTCCTTCAC-30). The PCR

fragments were inserted into the SmaI and EcoRV sites of

pBluescript SK(1), resulting in pUP31 and pUP32 plas-

mids, respectively. pUP3 was constructed by insertion of the

SphI–BamHI fragment of pUP31 into the corresponding

sites of pUP32. The nsoC gene was excised from pUP3 with

NdeI–SalI and ligated into the same sites of the pMHE6

expression vector (Fodor et al., 2004) (pUP3CFS).

For purification of rNsoC, a BL21(DE3)CodonPlus-RIL/

pUP3CFS culture (100 mL) induced with 0.5 mM isopropyl-

b-D-thiogalactopyranoside at 22 1C was harvested and re-

suspended in 9 mL of TBS buffer [50 mM Tris-HCl (pH 8.0),

150 mM NaCl, 2 mM dithiothreitol], and then sonicated.

Cell debris was removed by centrifugation (15 000 g, 10 min,

4 1C). The cell extract was loaded onto a gravity flow column

Fig. 1. (a) Organization of the genomic locus containing the nso operon in Thermococcus litoralis located upstream of the hyh2BDGA operon (both

gene clusters are deposited in the GenBank with the accession no.: EU024408). The main clones are shown by doublehead arrows. (b) Sequence of the

promoter region of the nso operon. The transcriptional start site is designated by a bold letter and an arrow. The TATA and transcription factor BRE

sequences, together with the putative ribosomal-binding site (RBS) of nsoA, are underlined. The translation start codon of nsoA is given in italics and the

first two amino acids of NsoA are also indicated.

FEMS Microbiol Lett 282 (2008) 8–14 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

9NADPH-dependent sulfur oxidoreductase from T. litoralis

containing 500 mL of Strep-Tactin Sepharose affinity resin

(IBA). The column was washed 10 times with 1.5 mL of TBS

buffer. The rNsoC was eluted three times with 300 mL TBS

supplemented with 2.5 mM desthiobiotin.

Enzyme assays

Sulfur reductase activity was routinely measured by the

polysulfide-dependent oxidation of NADPH. The assays

were performed at 65 1C in mixtures (2 mL) containing

100 mM HEPES buffer (pH 8.9), 0.3 mM NADPH, 0.2 mM

polysulfide and various amounts of samples. Activities were

calculated from the initial change in A340 nm, and were

corrected for nonenzymatic NADPH conversion. One unit

of sulfur reductase activity catalyzed the oxidation of

1mmol NADPH min�1. Polysulfide solution was prepared

by the reaction of 40 g of elemental sulfur with 20 g of

Na2S � 9H2O in 100 mL of water (Lengyel, 1999).

NADPH-dependent reduction of other substrates, such as

oxygen, benzyl viologen, methyl viologen and 5,50-dithio-

bis-(2-nitrobenzoic acid) (DTNB), was measured in mix-

tures containing 50 mM Tris-HCl (pH 8.0) and 0.3 mM

NADPH monitoring the absorbance changes at 340, 600

and 412 nm.

Glutamate dehydrogenase activity was determined at

65 1C by the glutamate-driven reduction of NADP1 mea-

sured at 340 nm (Ma et al., 1994).

Protein analysis

The native molecular mass (Mr) of rNsoC was determined

by gel filtration chromatography on a Bio-Silect SEC 250-5

size exclusion column (Bio-Rad) using thyroglobulin (Mr

670 000), bovine gamma globulin (Mr 158 000), ovalbumin

(Mr 44 000), myoglobin (Mr 17 000) and Vitamin B-12 (Mr

1350) as molecular mass standards.

For identification of the flavin cofactor in rNsoC by thin-

layer chromatography (TLC), it was extracted from the

protein as described in Stanton & Jensen (1993). The yellow

solution was run on a thin-layer silica gel plate

(5 cm� 10 cm, 200 mm) using an n-butanol–acetic acid–

water (12 : 3 : 5) solvent system. Pure flavin compounds:

FMN, FAD, and riboflavin were used as standards. After

drying the silica plates, the compounds were visualized by

UV shadowing (365 nm).

Results and discussion

Isolation of the nso operon

The operon encoding the soluble hydrogenase II (hyh2BG-

DA) in T. litoralis has been identified (A. Toth, M. Takacs, G.

Groma1, G. Rakhely & K.L. Kovacs, unpublished results).

Upstream of hyh2B, on a 7.9-kb genomic region (for

isolation see ‘Materials and methods’) five ORFs were

identified (Fig. 1a). Four of them formed a putative gene

cluster, which were designated as nso (NADPH-dependent

sulfur oxidoreductase). An additional ORF (orf1) with an

unknown function was identified downstream from the

nsoD gene, in the opposite direction.

Transcriptional analysis of the nso operon

The genomic organization of the nso genes suggested that

they form one transcription unit, which was confirmed by

RT-PCR experiments. cDNA was synthesized using a reverse

primer matching to the 30 end of the nsoD gene transcript,

and PCR was performed on this cDNA as a template with

primers designed for the nsoA gene. The expected PCR

product showed that full-length cDNA could be synthesized

containing even the nsoA gene; consequently, all the four nso

genes were located on a single transcript (data not shown).

Nevertheless, alternative promoters within the operon could

not be excluded.

The transcriptional start site of the nso genes was deter-

mined by primer-extension experiments (Fig. 1b). The initia-

tion point was preceded by typical archaeal promoter ele-

ments, like TATA box and transcription factor B recognition

element (BRE) located in appropriate positions (Soppa, 1999).

In silico analysis of Nso proteins

The deduced NsoA, NsoB and NsoC subunits showed the

highest resemblance to the products of putative genes of

Thermococcus kodakaraensis designated as TK1614, TK1613

and TK1612, but the nsoD gene was not found in the

T. kodakaraensis genome (Fukui et al., 2005) (Fig. 2). Gene

clusters organized similarly to the nso operon could not be

identified among other known archaeal sequences, indicat-

ing the narrow occurence of nso-like operons in the Archaea

domain.

The NsoA and NsoB proteins showed significant homol-

ogy to the NuoE and NuoF subunits of the NADH:ubiqui-

none oxidoreductase of E. coli (Leif et al., 1995) and to the

NuoE- and NuoF-related subunits of the NAD1-linked

formate dehydrogenase of Ralstonia eutropha (Oh & Bo-

wien, 1998), the [FeFe] hydrogenase of Thermotoga mariti-

ma (Verhagen et al., 1999), and the NADH dehydrogenase I

of Caldicellulosiruptor saccharolyticus encoded by the

Csac0619 gene (Fig. 2).

Two glycine-rich conserved regions were recognized in

NsoB. The GxGxxG motif is involved in the formation of the

hydrophobic ADP-binding pocket of NAD(P)(H)-binding

sites (McKie & Douglas, 1991). The presence of a glycine

residue in the last position of the motif is typical for

NAD(H)-binding proteins rather than NADP(H)-binding


10 A. Toth et al.

enzymes (Scrutton et al., 1990). The second motif is similar

to FMN-binding sites (Leif et al., 1995).

Because NsoA and NsoB exhibit high degrees of resem-

blance to the well-characterized NuoE and NuoF subunits of

various enzyme complexes, they are expected to have similar

functions, NADH oxidoreductase and electron transfer, in

the Nso complex proposed.

The NsoC protein showed similarity to a hypothetical

molybdopterin oxidoreductase from C. saccharolyticus

(Csac0621), to a monomeric [FeFe] hydrogenase of Desulfi-

tobacterium hafniense (Nonaka et al., 2006) and to a mono-

meric formate dehydrogenase of Shewanella oneidensis

(Heidelberg et al., 2002) (Fig. 2). A nearly 400 amino acid

long region of NsoC (and similar proteins) designated as the

GltD domain (c. residues 100� 500) resembled the small

subunit of eubacterial glutamate synthases (GltD) (Vanoni

& Curti, 1999). The GltD domain exhibited the highest

identity to GltD of Clostridium thermocellum (accession no.:

YP001036803) (Fig. 2) and showed similarity (45% identity)

to the large, GltD-like subunit of the sulfide dehydrogenase

of P. furiosus (Hagen et al., 2000).

In the GltD domain, a GxGxxA motif was suggested to be

responsible for NADP(H) rather than for NAD(H) binding

(Scrutton et al., 1990), and two other motifs were recognized

that corresponded to a FAD-binding site of some flavopro-

teins like disulfide reductases (Vanoni & Curti, 1999). The N

and C terminal parts of NsoC resembled the N terminus of

NuoG type proteins, which contained FeS cluster-binding

motifs of cysteines well characterized in NuoG of E. coli (Leif

et al., 1995) (Fig. 2). Furthermore, there are two conserved

sequence patterns located at the N terminus and close to the

C terminal end of the GltD domain characteristic exclusive

to NsoC-related proteins (Fig. 3).

The domain structure of NsoC and NsoC-related proteins

may have arisen from the integration of a eubacterial GltD

polypeptide into a eubacterial NuoG-related protein (Fig.

3). The proposed mechanism for the formation of these

fusion proteins could explain the arrangement of the

cysteine motifs present specifically in these enzymes.

The nsoD gene as part of the nso operon was found in

T. litoralis only (Fig. 2). The deduced NsoD showed the

highest sequence similarity to an uncharacterized NAD(P)H

Fig. 2. Schematic structural organization of Thermococcus litoralis Nso proteins and their homologues. Sequence motifs for binding of NADH, NADPH,

FMN, FAD, molybdopterin guanine dinucleotide (MGD) and H-cluster within the polypeptides are indicated. Black and gray bands represent sequence

motifs for the coordination of [2Fe–2S] and [4Fe–4S] centers, respectively. Light gray bands indicate sequence motifs characteristic of NsoC-type

proteins. Identities between Nso and its homologues are given above the schematic proteins. Identities of the GltD domain of NsoC with GltD domains

of NsoC-type proteins are indicated in brackets.



oxidase from P. furiosus (56% identity) (Robb et al., 2001).

However, its role in the Nso complex is cryptic.

Purification and characterization ofrecombinant NsoC protein

The in silico analysis of Nso proteins did not reveal sequence

motifs of known catalytic centers, like the H-cluster forming

residues characteristic of the [FeFe] hydrogenases or molyb-

dopterin-binding site peculiar to the formate dehydro-

genases. To establish the enzymatic properties of the Nso

complex, its simultaneous heterologous expression was

attempted. However, only NsoC could be expressed in

substantial amounts, while expression of the other subunits

was not successful. Reconstitution of the complex from the

individual components expressed separately also failed

because of poor expression of NsoA and NsoB. Therefore,

the 111-kDa recombinant NsoC protein (rNsoC) was ex-

pressed and purified by affinity chromatography. The native

molecular mass of purified protein, estimated by size-

exclusion chromatography, was 670 kDa, which suggested

that the native rNsoC was a hexamer.

In the UV-visible absorption spectrum of the oxidized

enzyme, the 390 nm peak indicated the possible presence of

iron–sulfur clusters (Orme-Johnson & Orme-Johnson,

1982), while the peak at 450 nm with a shoulder at around

480 nm was characteristic of oxidized flavoproteins (Fig. 4),

which agreed with the in silico analyis. The flavin cofactor

released after boiling of rNsoC in methanol was identified as

FAD by TLC (data not shown). The amount of FAD

extracted from rNsoC was 0.79� 0.02 mol mol�1 of enzyme,

assuming eFAD = 11.3 mM�1 cm�1 at 450 nm. Therefore,

rNsoC seems to contain one noncovalently bound FAD

cofactor per protein molecule.

The absorption peak at 450 nm decreased rapidly after

addition of NADPH under anaerobic conditions, indicating

the reduction of FAD. The peak partially reappeared when

oxygen was added to the enzyme solution (Fig. 4). These

observations showed that NsoC could use NADPH as an

electron donor and the FAD cofactor participated in the

electron transfer from NADPH to oxygen.

Enzymatic properties of rNsoC

Purified rNsoC catalyzed the oxidation of NADPH in the

presence of polysulfide with a specific activity of

0.35� 0.01 U mg�1; therefore, it was classified as sulfur

reductase. The enzyme could also catalyze the NADPH-

dependent reduction of artificial electron carriers, like

benzyl viologen and methyl viologen, and the disulfide

bond-containing substrate 5,50-dithiobis-(2-nitrobenzoic

acid) at rates of 17.11� 0.43, 1.17� 0.02 and

0.06� 0.002 U mg�1, respectively. Moreover, rNsoC had

remarkable NADPH oxidase activity under aerobic reaction

conditions (1.08� 0.07 U mg�1), which was also detected in

the case of some sulfur reductase enzymes, like sulfide

dehydrogenase of P. furiosus (Ma & Adams, 1994). In these

assays, NADH could not replace NADPH, which suggested

that rNsoC exclusively uses NADPH as an electron donor in

accordance with the in silico analysis. NADP1 reductase

reactions with reduced viologen dyes could not be detected.

The rate of the activity increased with the assay tempera-

ture up to 80 1C. However, the temperature optimum of the

enzyme could not be determined due to the significant

thermolability of NADPH above 80 1C. The pH optimum

of the sulfur reductase activity of rNsoC was around pH 8.9

(data not shown).

Fig. 3. Proposed mechanism for the formation of NsoC-type proteins.

Structural elements of the proteins are indicated as described in the

legend of Fig. 2.

Fig. 4. UV/visible absorption spectra of rNsoC. The absorption spectrum

of aerobically purified rNsoC (2.6 mg mL�1) in 50 mM Tris-HCl (pH 8.0)

and 150 mM NaCl at 65 1C is indicated by a solid line. The dashed line

indicates the spectrum after anaerobe addition of NADPH (1 mM), and

the dotted line shows the spectrum of rNsoC following the addition of

oxygen to the NADPH-supplemented samples.


12 A. Toth et al.

The kinetic parameters of rNsoC are summarized in

Table 1. The apparent Km value for polysulfide reduction

was slightly lower than that measured for P. furiosus enzymes

possessing sulfur reductase activity (Ma & Adams, 1994; Ma

et al., 2000). The affinity of rNsoC to O2 as another potential

physiological substrate was similar, but the NADPH oxidase

activity of the enzyme was much lower as compared with

that of NAD(P)H oxidases (Yang & Ma, 2005).

Enzymes that have sulfur or disulfide reductase activity

contain a single redox active cysteine or two cysteines in a

CxxC motif, which play an essential role in the catalytic

mechanism (Claiborne et al., 1999). However, a systematic

site-directed mutagenesis study revealed that in the se-

quence of NsoC no single cysteine was essential for catalytic

function (data not shown). This indicates a potentially new,

although unknown, catalytic mechanism.

Sulfur reductase activity in T. litoralis

To demonstrate that T. litoralis cells have NAD(P)H-depen-

dent sulfur reductase activity, cell extracts were prepared,

which catalyzed the reduction of colloidal sulfur and poly-

sulfide using NADPH as the electron donor at a rate of

0.021 U mg�1 and 0.065 U mg�1, respectively. These values

were similar to the sulfur reductase activities of P. furiosus

cell extracts (Ma & Adams, 1994). NADH-dependent poly-

sulfide reduction with T. litoralis cell extracts could also be

measured (specific activity of 0.029 U mg�1). To localize the

sulfur reductase activity of T. litoralis, cell fractions were

prepared (120 000 g for 1.5 h). Ninety-two percent and 74%

of the NADPH-dependent colloidal sulfur and polysulfide

reductase activities were present in the cytoplasmic frac-

tions, respectively. A similar distribution was found for the

marker cytoplasmic enzyme, glutamate dehydrogenase

(GDH). These results suggested the presence of cytoplasmic

NADPH-dependent sulfur reductase in T. litoralis.

Conclusions

An operon consisting of four genes (nsoABCD) was isolated

from T. litoralis. A similar gene cluster could be found only

in the genome of T. kodakaraensis among the sequenced

Archaea species. The operon codes for a complex in which

NsoABC subunits seem to be more closely related to various

eubacterial nucleotide-binding oxidoreductases than to ar-

chaeal proteins; therefore, this gene cluster is likely of

bacterial origin. This hypothesis is supported by the fact

that the codon frequency in nsoABC genes does not corre-

spond to the overall codon usage of T. litoralis (http://

www.kazusa.or.jp/codon/).

The complex had an unusual hybrid domain structure

and no typical catalytic center could be recognized. In silico

analysis suggested the complex to bind both NADH and

NADPH, flavins and iron–sulfur clusters. Biochemical ana-

lysis showed that the recombinant NsoC subunit had

NADPH-dependent sulfur reductase activity. According to

in silico analysis, the NsoB subunit can bind NADH; there-

fore, both NADH and NADPH might serve as substrates for

the Nso complex. Moreover, oxygen could also be used as a

substrate; therefore, the enzyme might have a role in the

regeneration of NADP1 pool via sulfur reduction and in the

protection of cells against oxidative stress.

Acknowledgements

This work has been supported by EU FP6 projects (HyVolu-

tion SES6 019825 and NEST STRP SOLAR-H 5166510) and

by domestic funds (NKTH, GVOP, Asboth, Baross, DEAK-

KKK, KN-RET). We gratefully acknowledge Rozsa Verebely

for excellent technical assistance.

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Table 1. Kinetic parameters for rNsoC�

Substrate (mM) Apparent Km (mM) Apparent Vmax (U mg�1)

Polysulfide (0–0.9) 0.54� 0.08 1.39� 0.17

DTNBw (0–4.0) 0.55� 0.09 0.18� 0.03

Benzyl viologen (0–0.3) 0.056� 0.003 28.02� 2.04

Methyl viologen (0–2.0) 0.84� 0.14 9.43� 0.66

Oxygen (0–0.134) 0.11� 0.02 1.81� 0.17

NADPHz (0–0.3) 0.087� 0.008 1.34� 0.09

�Assays were carried out at 65 1C using 0.3 mM NADPH as a cosubstrate.

The experimental data were curvefitted by nonlinear regression.w5,50-Dithiobis-(2-nitrobenzoic acid).zThe cosubstrate was oxygen under aerobic reaction conditions.



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A novel NADPH-dependent oxidoreductase with a unique domain structure in the hyperthermophilic Archaeon, Thermococcus litoralis