Experimental results showed that NaoA can con-vert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds.. NADH and nitro blue tetrazolium are strong i
Trang 1Cloning, expression and characterization of a gene encoding
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-genes7100 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 48–56C, respectively The Kmof NaoA for nitroethane is
26.8 mM 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 saturmus var 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 saturmus var mrakii and nitroalkane oxidase were cloned and expressed in Escherichia coli [14,15]
Dhawale et al [16] reported that crude cell-free extracts of Streptomycescould 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 PTH270, 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 saturmus var 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
M A T E R I A L S A N D M E T H O D S
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 28C [22] JMl09 and BL21(DE3) strains were grown at 37C, in Luria–Bertani medium supplemented with100 lgÆmL)1 ampicillin when necessary [23]
DNA manipulations Plasmid and chromosomal DNA was isolated from Streptomycesor 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: tanhr@sun.im.ac.cn
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: Department of Biology, University of Waterloo,
Waterloo, ON N2L 3G1 Canada.
(Received 7 August 2002, revised 25 October 2002,
accepted 5 November 2002)
Trang 2Transformation 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 withexonuclease III by the reported method
[24] to generate a set of nested deletions from eachend
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-32P]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 BasicBLAST
search[26]
Primers and PCR conditions
To achieve overexpression of naoA, two primers were
designed from the complete DNA sequence of naoA (P1,
5¢-GACATATGTCCTCCGCGCTGA-3¢; P2, 5¢-GGAA
GCTTTCACCCCTTACGGGA-3¢, with NdeI and HindIII
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 95C for 5 min
followed by 30 cycles of amplification (95C for 1 min,
55C for 1 min, and 72 C for 1 min) and an additional
extension step at 72C for 10 min The PCR product
(about 1.1 kb) was purified by agarose gel electrophoresis
and then subcloned into the NdeI and HindIII 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 coliBL21(DE3) containing plasmid pNA101 was grown
at 37C for 12–14 hor overnight in Luria–Bertani
medium supplemented withampicillin (100 lgÆmL)1), and
then 40 mL Luria–Bertani medium was inoculated with
40 lL of the above fresh overnight culture and incubated
at 37C withshaking until cells were grown to D600¼ 0.4,
usually about 2.5–3 h The culture was induced (1 mM
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 mM 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 mM nitroalkane, cell extract and 0.1M 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 withMBTH using an improved method [29,30] The reaction of the aldehyde group withferric 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 mM 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 for 30 min and th en dissolved in 10 mMsodium phosphate buffer (pH 8.0)
Step 2: The crude protein solution was loaded onto a Sephadex G75 column pre-equilibrated with 10 mMsodium 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 0.2M Th en, th e protein solution was run on a DEAE-Sepharose Fast Flow column pre-equili-brated with10 mMsodium phosphate buffer (pH 8.0) The column was washed with 0.2MNaCl until no protein was eluted, and then bound NaoA was eluted with 0.35MNaCl
in th e same buffer
Step 4: After being desalted withan ultrafiltration tube (Pall Corporation), the active fractions were further run on
a DEAE-Sepharose Fast Flow column pre-equilibrated with10 mM sodium phosphate buffer (pH 8.0) The column was first washed with the same buffer containing 0.2M NaCl, and then the proteins were eluted with a 0.2–0.4M NaCl gradient in the above buffer (flow rate, 0.4 mLÆmin)1)
Trang 3Experiments on NaoA properties
The enzymatic reaction was assayed in sodium phosphate
buffer (0.1M) 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] Kmand Vmaxof purified NaoA were determined
from a double-reciprocal plot according to the
Lineweaver-Burk equation [32]
R E S U L T S
Cloning of 1.5-kb DNA fragment
A 320-bp DNA fragment located upstream of PTH270
(a differentiation-related promoter of Streptomyces
coeli-color) was obtained by digesting pIJ4477 with SmaI and
HindIII This DNA fragment was labeled with the
digoxigenin-11-dUTP kit (Roche, Mannheim, Germany)
and used as a probe for Southern-blot hybridization
withthe digested genomic DNA of S ansochromogenes
Approximately 7.0-kb DNA fragments witha 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 with1092
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% withthe putative oxidoreductase from
Streptomyces coelicolor, 27% with2-nitropropane
dioxyg-enase of W saturmus var mrakii, 26% with2-nitropropane
dioxygenase of N crassa, and 36% withthe 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 ofnaoA 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 withnitroethane, 1-nitropropane and 2-nitropropane were, respectively, 100%, 90.7%, 5.89% when the substrate concentration was 4 mM
Reaction mixtures containing the purified NaoA and substrate (1-nitropropane, 2-nitropropane or nitroethane) were incubated for 5 min at 37C 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 withhighiden-tity are shaded The program OMIGA 2.0 was used to compare amino-acid sequences.
Trang 42-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 withMBTH After reaction withMBTH, 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 withFeCl3
using nitroethane or 1-nitropropane as substrate (maximum
peak 640–670 nm) This result showed that the
correspond-ing aldehydes were formed durcorrespond-ing 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+) and m/z 43 consistent withthose
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 48–56C, respectively, in 0.1M sodium phosphate buffer NaoA activity increased over the temperature range 20–50C but declined rapidly above 60C
The effects of various compounds on NaoA activity were also examined (Table 2) Mn2+ increased the enzyme activity slightly, and Cu2+inhibited it Mg2+and Ca2+ 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 mM When the amount of superoxide dismutase reached 200 U, the relative activity of NaoA remained 6.1% and 51%, respectively, with2-nitropropane and nitroethane
as substrate These results suggest that superoxide anion radicals are essential intermediates in the oxidation of nitroalkane by NaoA
The Kmof purified NaoA for nitroethane was found to be
26.8 mM, and Vmax for the formation of nitrite 0.175 lmolÆmin)1Ælg)1according to the Lineweaver-Burk equation
D I S C U S S I O N
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 Streptomyceshas 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, 97 kDa; BSA, 66 kDa;
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, subsub-strate nitroethane; lane 3, subsub-strate 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 (%)
Trang 5GXGXXA, 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 Km of NaoA for
nitroethane (26.8 mM) is similar to that of 2-nitropropane
dioxygenase [4,6], but is quite different from that of
nitroalkane oxidase from F oxysporum (1 mM) [8] From
the substrate specificity, 2-nitropropane is the preferred
substrate for 2-nitropropane dioxygenase from W
satur-mus var mrakii [4] and N 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], whichis consistent withthe
reaction mechanism for 2-nitropropane denitrification to
acetone proposed by Gorlatova et al [6] and Kuo &
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 Mg2+and Ca2+did not affect NaoA activity, and are therefore probably not necessary for the oxidation However, Cu2+ strongly inhibited the activity of NaoA, and Mn2+slightly increased the activity This implies that there may be a Mn2+or Cu2+
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 withthose 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 polluapplica-tion 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
A C K N O W L E D G E M E N T S
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
Relative activity (%)
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