Báo cáo Y học: Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes pot

Cloning, expression and characterization of a gene encoding
nitroalkane-oxidizing enzyme from
Streptomyces ansochromogenes
Jihui Zhang, Wenbo Ma* and Huarong Tan
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
A nitroalkane-oxidizing enzyme gene (naoA) was cloned
from a genomic DNA library of Streptomyces ansochromo-
genes 7100. The deduced protein (NaoA) of this gene con-
tains 363 amino acids and has high similarity to several
nitroalkane-oxidizing enzymes from various micro-organ-
isms. The naoA gene was subcloned into an expression vector
pET23b and overexpressed in Escherichia coli BL21(DE3).
The protein was then purified, and its characteristics were
studied. Experimental results showed that NaoA can con-
vert 1-nitropropane, 2-nitropropane and nitroethane into
the corresponding carbonyl compounds. The optimal pH
and temperature for NaoA was found to be pH 7–8 and
48–56 °C, respectively. The K
m
of NaoA for nitroethane is
 26.8 m
M
. NADH and nitro blue tetrazolium are strong
inhibitors of NaoA, and thiol compounds and superoxide
dismutase partially inhibit the enzyme activity. Therefore,
superoxide may be an essential intermediate in the oxidation
of nitroalkane by NaoA.
Keywords: enzymatic properties; expression; gene cloning;
nitroalkane-oxidizing enzyme; Streptomyces.
Nitroalkane compounds are widely used in chemical indus-
try as intermediates, solvents and fuel for rockets [1] and are

released in large quantities into the environment. Mean-
while, certain micro-organisms and many leguminous plants
produce nitroalkane compounds [2]. These materials are
hazardous and can result in environmental contamination.
So the conversion of nitro groups by biocatalysts is useful in
industry as well as in environmental conservation. Enzymes
that can convert nitroalkanes into less harmful species have
been purified and characterized from micro-organisms. They
include 2-nitropropane dioxygenase from Williopsis satur-
mus var. mrakii [3–5] and Neurospora crassa [6,7] and
nitroalkane oxidase from Fusarium oxysporum [8] and
Aspergillus flavus [9]. The conversion of nitro compounds
into less harmful materials by nitroalkane-oxidizing enzymes
in organisms may have the physiological significance of
inactivating the natural defenses of plants [10]. The reaction
mechanisms of several nitroalkane-oxidizing enzymes have
been analyzed [6,11,12]. Thus, 2-nitropropane dioxygenase
from W.saturmusvar. mrakii catalyzes the incorporation of
two atoms of oxygen molecule into two molecules of the
same acceptor, and the enzyme is an intermolecular dioxyg-
enase [4], and nitroalkane oxidase from F. oxysporum has a
hydrophobic microenvironment of the flavin cofactor
[11,13]. The genes encoding 2-nitropropane dioxygenase
from W.saturmusvar. mrakii and nitroalkane oxidase were
cloned and expressed in Escherichia coli [14,15].
Dhawale et al. [16] reported that crude cell-free extracts of
Streptomyces could catalyze the oxidation of nitroalkanes to
form carbonyl compounds and nitrite, but the genes related
to these enzymes in Streptomyces have not been reported so
far. Our previous experiments revealed that DNA upstream

of P
TH270
, a differentiation-related promoter in Streptomyces
[17,18], contained an incomplete ORF the deduced product
of which had high similarity to 2-nitropropane dioxygenase
from W.saturmusvar. mrakii. This led to the experiment
to identify whether the protein encoded by the complete
DNA fragment can catalyze the oxidation of nitroalkanes.
In this paper, we describe the cloning and characterization of
a novel gene (naoA) that encodes nitroalkane-oxidizing
enzyme in S. ansochromogenes.
MATERIALS AND METHODS
Strains, plasmids and growth conditions
S. ansochromogenes 7100 [19], E. coli JMl09, BL21(DE3)
[20], pBluescript Ml3
–
, pET23b (Novagen) and M13 KO7
[21] as the helper phage were collected in this laboratory.
pIJ4477 was constructed during the work described in [18];
pTH1104 (Ml3
–
containing naoA) and pNA101 (pET23b
containing naoA) were constructed in this work. S. anso-
chromogenes mycelium was grown in yeast extract/malt
extract liquid medium on a rotary shaker at 28 °C[22].
JMl09 and BL21(DE3) strains were grown at 37 °C, in
Luria–Bertani medium supplemented with 100 lgÆmL
)1
ampicillin when necessary [23].
DNA manipulations

Plasmid and chromosomal DNA was isolated from
Streptomyces or E. coli by established techniques [22,23].
Correspondence to H. Tan, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100080, China.
Fax: + 86 10 62654083, Tel.: + 86 10 62654083,
E-mail:
Abbreviations: naoA, nitroalkane-oxidizing enzyme gene A; MBTH,
3-methyl benzothiazolone hydrazone hydrochloride.
Note: the nucleotide sequence of naoA gene has been deposited in
GenBank under the accession number AF284037.
*Present address:DepartmentofBiology,UniversityofWaterloo,
Waterloo, ON N2L 3G1. Canada.
(Received 7 August 2002, revised 25 October 2002,
accepted 5 November 2002)
Eur. J. Biochem. 269, 6302–6307 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03350.x
Transformation of E. coli strains, Southern blotting, and
colony hybridization were carried out as described by
Sambrook et al. [23]. Restriction enzymes and T4 DNA
ligase were purchased from Boehringer-Mannheim and
Sino-American Biotechnology Company (Luoyang, Chi-
na)
1
. DIG labeling and detection kits (Boehringer-Mann-
heim) were used for preparation of DNA probes according
to the protocols of the manufacturer.
DNA sequencing and analysis
Plasmid pTH1104 containing the target fragment was
digested with exonuclease III by the reported method
[24] to generate a set of nested deletions from each end
of the inserts. Appropriately deleted derivatives were

sequenced by the dideoxy chain termination method
using the Taq Track sequencing kit (Promega, Madison,
WI, USA) and [a-
32
P]dCTP as the labeled nucleotide.
ORF analysis was based on the specific codon usage of
Streptomyces [25]. Deduced amino-acid sequence was
compared with the database in the National Center for
Biotechnology Information (NCBI) using the Basic
BLAST
search [26].
Primers and PCR conditions
To achieve overexpression of naoA, two primers were
designed from the complete DNA sequence of naoA (P1,
5¢-GA
CATATGTCCTCCGCGCTGA-3¢;P2,5¢-GGAA
GCTTTCACCCCTTACGGGA-3¢,withNdeIandHindIII
restriction sites underlined). Pfu DNA polymerase (Sangon
Co., Shanghai, China) was used to amplify naoA,with
pTH1104 as template. The following PCR program was
performed: an initial denaturalization at 95 °Cfor5min
followed by 30 cycles of amplification (95 °Cfor1min,
55 °C for 1 min, and 72 °C for 1 min) and an additional
extensionstepat72°C for 10 min. The PCR product
(about 1.1 kb) was purified by agarose gel electrophoresis
and then subcloned into the NdeIandHindIII sites of
pET23b for overexpression.
Electrophoresis of proteins
SDS/PAGE was carried out as described previously [23].
IEF was performed using a Computer Controlled Electro-

phoresis Power Supply and a Mini IEF Cell (Bio-Rad).
Protein standards of different pI were as follows: amylo-
glucosidase, pI 3.6; b-lactoglobulin A, pI 5.1; myoglobin, pI
6.8/7.2; trypsinogen, pI 9.3. The pI of NaoA was determined
from a standard curve of pI and migration distance (cm) of
protein standards.
Enzyme assay and analytical methods
E. coli BL21(DE3) containing plasmid pNA101 was grown
at 37 °C for 12–14 h or overnight in Luria–Bertani
medium supplemented with ampicillin (100 lgÆmL
)1
), and
then 40 mL Luria–Bertani medium was inoculated with
40 lL of the above fresh overnight culture and incubated
at 37 °C with shaking until cells were grown to D
600
¼ 0.4,
usually about 2.5–3 h. The culture was induced (1 m
M
isopropyl thio-b-
D
-galactoside) and grown for a further
3 h, and then the cells were harvested by centrifugation at
10 000 g for 3 min and suspended in 100 m
M
sodium
phosphate buffer (pH 8.0). A 10-mL volume of cell
suspension was discontinuously sonicated (100 W; JY96-
II sonicator) for 5 min on ice to generate cell extracts after
centrifugation at 10 000 g for 3 min. Protein concentra-

tions were determined by the Biuret reaction using BSA as
standard [27]. Activity of NaoA was detected with
nitroethane, 1-nitropropane, 2-nitropropane or nitrometh-
ane as substrate. The standard reaction mixture consisted
of 4 m
M
nitroalkane, cell extract and 0.1
M
sodium
phosphate (pH 8.0 for 2-nitropropane and pH 7.0 for 1-
nitropropane or nitroethane) and was finally maintained in
a 0.6-mL volume. Nitrite released from the reaction was
determined by the method of Little [28]. One unit of NaoA
is defined as the amount of enzyme required to catalyze the
formation of 1 lmol nitriteÆmin
)1
. Carbonyl compounds
(aldehyde and ketone) can react with 3-methyl benzothi-
azolone hydrazone hydrochloride (MBTH; purchased
from the Fluka Chemical Company) to form azines, which
display a characteristic absorbance peak at 304–310 nm.
Therefore, formation of carbonyl compounds from nitro-
alkanes can be demonstrated with MBTH using an
improved method [29,30]. The reaction of the aldehyde
group with ferric chloride was used to detect them during
this study, and acetone was determined with GC-MS on
the Shimadzu GCMS-QP5050A. Acetaldehyde and acet-
one were used as standards.
Purification of NaoA
Cells of BL21(DE3) carrying pNA101 were harvested by

centrifugation (10 000 g, 3 min). After suspension in
100 m
M
sodium phosphate buffer (pH 8.0), they were
sonicated and centrifuged at 10 000 g for 30 min to remove
cell debris. NaoA was purified from the supernatant
according to the following steps.
Step 1: Solid ammonium sulfate was added to the crude
extracts to a final concentration of 10% saturation, and the
precipitate was removed by centrifugation. Then, solid
ammonium sulfate was added to the supernatant to give
80% saturation. The resulting precipitate containing the
enzyme activity was collected by centrifugation at 10 000 g
for30minandthendissolvedin10m
M
sodium phosphate
buffer (pH 8.0).
Step 2: The crude protein solution was loaded onto a
Sephadex G75 column pre-equilibrated with 10 m
M
sodium
phosphate buffer (pH 8.0) and eluted with the same buffer.
The active fractions were collected.
Step 3: NaCl was added to the pooled active fractions to a
final concentration of 0.2
M
.Then,theproteinsolutionwas
run on a DEAE-Sepharose Fast Flow column pre-equili-
brated with 10 m
M

sodium phosphate buffer (pH 8.0). The
column was washed with 0.2
M
NaCl until no protein was
eluted, and then bound NaoA was eluted with 0.35
M
NaCl
inthesamebuffer.
Step 4: After being desalted with an ultrafiltration tube
(Pall Corporation), the active fractions were further run on
a DEAE-Sepharose Fast Flow column pre-equilibrated
with 10 m
M
sodium phosphate buffer (pH 8.0). The
column was first washed with the same buffer containing
0.2
M
NaCl, and then the proteins were eluted with a
0.2–0.4
M
NaCl gradient in the above buffer (flow rate,
0.4 mLÆmin
)1
).
Ó FEBS 2002 A nitroalkane-oxidizing enzyme of Streptomyces (Eur. J. Biochem. 269) 6303
Experiments on NaoA properties
The enzymatic reaction was assayed in sodium phosphate
buffer (0.1
M
) at different pH values and temperatures to

define optimal conditions. Various compounds were also
investigated for their inhibitory effects on enzyme activity.
Superoxide dismutase was purchased from Sigma Chemical
Company; its unit of activity was as defined by the
manufacturer. Different concentrations of nitroethane were
used to test the relationship between initial velocity and
substrate concentration. Velocity was determined by
detecting the formation of nitrite using the method of Ida
et al.[31].K
m
and V
max
of purified NaoA were determined
from a double-reciprocal plot according to the Lineweaver-
Burk equation [32].
RESULTS
Cloning of 1.5-kb DNA fragment
A 320-bp DNA fragment located upstream of P
TH270
(a differentiation-related promoter of Streptomyces coeli-
color) was obtained by digesting pIJ4477 with SmaIand
HindIII. This DNA fragment was labeled with the
digoxigenin-11-dUTP kit (Roche, Mannheim, Germany)
and used as a probe for Southern-blot hybridization
with the digested genomic DNA of S. ansochromogenes.
Approximately 7.0-kb DNA fragments with a positive
signal were separated from the genomic DNA digested
with NotI by agarose gel electrophrosis, and then a
partial DNA library was constructed in E. coli JM109
using pBluescript M13

–
as vector. The library was
screened by colony hybridization using the above probe.
Several positive colonies were identified and confirmed
by Southern-blot hybridization (data not shown). The
recombinant plasmid was further digested with SstII, and
a 1.5-kb DNA fragment still displayed a strong positive
signal after hybridization.
DNA sequencing analysis
DNA sequencing analysis showed that the 1.5-kb DNA
fragment contains one complete ORF with 1092 nucleo-
tides. The overall G + C content is 74%, which is typical
for genes of Streptomyces. A potential ribosome-binding site
(GGAAGGA) was located at the 18–24 base positions from
the start codon (ATG). The deduced protein had a
molecular mass of  37 kDa and showed identity (Blast
output) with the following proteins in database searches
(Fig. 1): 78% with the putative oxidoreductase from
Streptomyces coelicolor, 27% with 2-nitropropane dioxyg-
enase of W.saturmusvar. mrakii, 26% with 2-nitropropane
dioxygenase of N. crassa, and 36% with the putative
2-nitropropane dioxygenase encoded by yrpB of Bacillus
subtilis. Therefore, the gene product may be involved in the
degradation of nitroalkanes and this gene was designated
naoA (nitroalkane-oxidizing enzyme gene).
Expression of
naoA
in
E. coli
To study the function of the naoA gene, it is necessary to

obtain an adequate amount of NaoA protein. Therefore,
the naoA gene was subcloned into pET23b to generate
plasmid pNA101, and then it was introduced into
BL21(DE3) for high-level expression under the control of
the T7 promoter. After induction with isopropyl thio-
b-
D
-galactoside, a 37-kDa protein band from the extracts of
BL21(DE3)/pNA101 appeared on SDS/PAGE, whereas no
protein bands from the extracts of BL21(DE3)/pET23b as
control were found at the same position on SDS/PAGE
(Fig. 2). The result indicated that the naoA gene was
efficiently expressed in E. coli.
Purification and characterization of NaoA
Protein extracts of BL21(DE3)/pNA101 were separated by
gel filtration, anion-exchange column chromatography, and
ultrafiltration. The purified NaoA was further detected by
SDS/PAGE (Fig. 2). The data for NaoA purification are
summarized in Table 1. The specific activity of the purified
NaoA was about 21 times higher than that of crude extract,
and the yield was 34%. The relative activities of purified
NaoA with nitroethane, 1-nitropropane and 2-nitropropane
were, respectively, 100%, 90.7%, 5.89% when the substrate
concentration was 4 m
M
.
Reaction mixtures containing the purified NaoA and
substrate (1-nitropropane, 2-nitropropane or nitroethane)
were incubated for 5 min at 37 °C. After the reaction
solution was mixed with o-aminophenylsulfuric acid and

a-naphthanamine solutions, a red color appeared. The
cell extracts of BL21(DE3)/pET23b as control did not
show a coloring reaction, indicating that 1-nitropropane,
Fig. 1. Comparison of NaoA with other nitroalkane-oxidizing enzymes.
SA, NaoA from S. ansochromogenes; SC, putative 2-nitropropane
dioxygenase from S. coelicolor; WS, 2-nitropropane dioxygenase from
W.saturmus var. mrakii; NC, 2-nitropropane dioxygenase from
N. crassa; BS, 2-nitropropane dioxygenase-related protein encoded by
yrpB gene from Bacillus subtilis. Amino-acid residues with high iden-
tity are shaded. The program
OMIGA
2.0 was used to compare amino-
acid sequences.
6304 J. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
2-nitropropane and nitroethane can be oxidized and deni-
trified to form nitrite by NaoA. Furthermore, it was clear
that nitromethane was not a substrate of NaoA because no
red color, which would have indicated the production of
nitrite, was seen in assays containing nitromethane (Fig. 3).
In addition to nitrite formation, carbonyl compounds
released in the oxidation of nitroalkanes by NaoA were
determined with MBTH. After reaction with MBTH, the
UV spectra of the reaction solutions with nitroethane,
1-nitropropane or 2-nitropropane had a typical maximum
absorption at 304–310 nm, which was identical with that of
the expected carbonyl products reacted with MBTH. A
deep green color was obtained after reaction with FeCl
3
using nitroethane or 1-nitropropane as substrate (maximum
peak 640–670 nm). This result showed that the correspond-

ing aldehydes were formed during the oxidation of nitro-
ethane or 1-nitropropane in the presence of NaoA. When
2-nitropropane was used as substrate, the carbonyl com-
pound formed in the reaction solution was further con-
firmed to be acetone by its mass spectrum, which displayed
fragments of m/z 58 (M
+
)andm/z 43 consistent with those
of acetone standard.
Properties of NaoA
The pI of NaoA is about 5.2 according to the standard plot
between the protein’s pI and its migration distance (cm) in
IEF. The optimal pH and temperature of purified NaoA
were 7.0–8.0 (data not shown) and 48–56 °C, respectively, in
0.1
M
sodium phosphate buffer. NaoA activity increased
over the temperature range 20–50 °C but declined rapidly
above 60 °C.
The effects of various compounds on NaoA activity were
also examined (Table 2). Mn
2+
increased the enzyme
activity slightly, and Cu
2+
inhibited it. Mg
2+
and Ca
2+
did not affect NaoA activity. Thiol groups may be involved

in the active site of NaoA because thiol compounds
(2-mercaptoethanol, GSH) partially inhibited activity.
Unlike the nitroalkane oxidase from F. oxysporum [8],
NADH strongly decreased the NaoA activity. NaoA is
almost completely inactive in the presence of the super-
oxide-scavenging agent nitro blue tetrazolium at a concen-
tration of 5 m
M
. When the amount of superoxide dismutase
reached 200 U, the relative activity of NaoA remained 6.1%
and 51%, respectively, with 2-nitropropane and nitroethane
as substrate. These results suggest that superoxide anion
radicals are essential intermediates in the oxidation of
nitroalkane by NaoA.
The K
m
of purified NaoA for nitroethane was found to be
 26.8 m
M
,andV
max
for the formation of nitrite
0.175 lmolÆmin
)1
Ælg
)1
according to the Lineweaver-Burk
equation.
DISCUSSION
We have cloned and determined the complete sequence of a

gene encoding nitroalkane-oxidizing enzyme from Strepto-
myces; partial purification of the related enzyme from
Streptomyces has been reported [16]. The deduced amino-
acid sequence of NaoA from S. ansochromogenes has high
identity with that of the putative oxidoreductase from
S. coelicolor [33,34]. The two proteins consist of 363 and 364
residues, respectively. Moreover, the consensus sequence
Fig. 2. SDS/PAGE of NaoA expressed in E. coli and its purification.
Lane 1, total protein from BL21(DE3)/pET23b; lane 2, total protein
from strain BL21(DE3)/pNA101; lane 3, recombinant NaoA after
80% ammonium sulfate fraction; lane 4, recombinant NaoA after
Sephadex G75 chromatography; lane 5, purified recombinant NaoA
after DEAE-Sepharose Fast Flow chromatography; lane 6, standard
molecular mass markers (phosphorylase b,97kDa;BSA,66kDa;
ovalbumin, 45 kDa).
Fig. 3. Assay of NaoA activity. (A) Protein extracts from BL21(DE3)/
pET23b. (B) Protein extracts from BL21(DE3)/pNA101; lane 1, sub-
strate 2-nitropropane; lane 2, substrate nitroethane; lane 3, substrate
1-nitropropane; lane 4, substrate nitromethane. 2 m
M
substrate and
100 lL protein extracts were used in the reaction.
Table 1. Purification of NaoA from E. coli. The activity is measured according to the formation of nitrite using 1-nitropropane as substrate.
Purification step
Total protein
(mg)
Total activity
(U)
Specific activity
[UÆ(mg protein)

)1
]
Purification
(fold)
Yield
(%)
Cell extract 307 1860 6 1.00 100
Sephadex G75 172 1756 10 1.7 94
DEAE-Sepharose Fast Flow 5 633 127 21 34
Ó FEBS 2002 A nitroalkane-oxidizing enzyme of Streptomyces (Eur. J. Biochem. 269) 6305
GXGXXA, which exists in many nucleotide-binding
domains of dehydrogenases [35], was found at positions
36–41 in the deduced NaoA protein (GSGFLA) as well as
in the putative oxidoreductase of S. coelicolor (GLGFLA).
NaoA also displayed features that resemble those of
2-nitropropane dioxygenase from W. saturmus var. mrakii
and those of nitroalkane oxidase from F. oxysporum. Both
carbonyl compounds and nitrite, the common products of
nitroalkane oxidation catalyzed by 2-nitropropane dioxyg-
enase and nitroalkane oxidase, were detected in the
oxidation of 1-nitropropane, 2-nitropropane and nitro-
ethane catalyzed by NaoA, indicating that NaoA is a type
of nitroalkane-oxidizing enzyme. Furthermore, the deduced
amino-acid sequence of NaoA has higher identity with
those of 2-nitropropane dioxygenase characterized in
W. saturmus var. mrakii [14], and the inhibitory effects
of various compounds on NaoA activity are also similar to
2-nitropropane dioxygenase. Therefore, NaoA is possibly
a nitroalkane dioxygenase-like enzyme. The enzymatic
properties of NaoA are a little different from those of

other nitroalkane dioxygenases. The K
m
of NaoA for
nitroethane (26.8 m
M
) is similar to that of 2-nitropropane
dioxygenase [4,6], but is quite different from that of
nitroalkane oxidase from F. oxysporum (1 m
M
)[8].From
the substrate specificity, 2-nitropropane is the preferred
substrate for 2-nitropropane dioxygenase from W.satur-
mus var.mrakii[4]andN. crassa [6]. However, NaoA
activity is much higher with 1-nitropropane and nitro-
ethane than with 2-nitropropane.
We report some of the basic properties of NaoA. In the
nitroalkane oxidation reaction, the superoxide anion is an
essential intermediate [36]. On the basis of the inhibitory
effects of various compounds on 2-nitropropane dioxyge-
nase and by adding superoxide anion to the reaction
mixture to induce nitroalkane oxygenation, it was demon-
strated that superoxide anion indeed participates in the
reaction as an intermediate [36], which is consistent with the
reaction mechanism for 2-nitropropane denitrification to
acetone proposed by Gorlatova et al.[6]andKuo&
Fridovich [37]. In this study, NaoA activity was strongly
inhibited by superoxide anion radical scavengers (nitro blue
tetrazolium and NADH) as well as superoxide dismutase,
which is in accord with the above 2-nitropropane dioxyg-
enase [6,36], confirming that the superoxide anion is an

essential intermediate in the nitroalkane oxidation catalyzed
by NaoA. In addition, Gadda et al. [10,38] demonstrated
that a cysteine residue and a neighboring tyrosine residue
were present in the active site of the flavoprotein nitroalkane
oxidase from F. oxysporum, whereas these two amino acids
were not conserved in NaoA from S. ansochromogenes and
the putative oxidoreductase from S. coelicolor. Possibly,
NaoA uses another catalytic pathway. Mg
2+
and Ca
2+
did
not affect NaoA activity, and are therefore probably not
necessary for the oxidation. However, Cu
2+
strongly
inhibited the activity of NaoA, and Mn
2+
slightly increased
the activity. This implies that there may be a Mn
2+
or Cu
2+
binding site in the enzyme; metal ions may also act in other
ways.
We conclude that NaoA is a nitroalkane dioxygenase-like
enzyme rather than a nitroalkane oxidase. Its characteristics
are not identical with those of any reported nitroalkane-
oxidizing enzymes, therefore it may be a novel enzyme able
to convert nitroalkanes into the corresponding carbonyl

compounds. These studies provide the basis for its applica-
tion in the treatment of environmental pollution by certain
chemicals. Many nitro group compounds are released into
the environment, many of which have strong mutagenic
activity [39]. They may be absorbed through food and water
resulting in serious diseases. Therefore, biodegradation of
nitro group compounds is very important to environmental
conservation.
ACKNOWLEDGEMENTS
This work was supported by grants from the National Natural Science
Foundation of China (Grant nos. 39830010 and 39925002) and the
National ‘863’ Plan Programme of China (contract no.
2001AA214071). We are grateful to Professor Keith Chater (John
Innes Center, Norwich, UK) for critical reading and help in preparation
of this paper.
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Table 2. Effects of different compounds on NaoA activity. The different compounds were added to the NaoA reaction solution and the reaction was
carried out under standard conditions (pH 7.0, 37 °C for 5 min). The activity is measured according to the formation of nitrite using 1-nitro-

propane as substrate. NBT, Nitro blue tetrazolium; SOD, superoxide dismutase.
Compound
Relative
activity (%) Compound
Relative
activity (%)
Control 100 2-Mercaptoethanol (2 m
M
) 15.5
MnCl
2
(1 m
M
) 117.8 GSH (2 m
M
) 37.9
CuSO
4
(1 m
M
) 16.9 NADH (2 m
M
)0
CaCl
2
(1 m
M
) 82.0 EDTA (2 m
M
) 74.6

MgSO
4
(1 m
M
) 89.8 NBT (5 m
M
)0
NAD (2 m
M
) 92.5 SOD (100 U) 64.0
Cystine (2 m
M
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