stenotrophomonas sp. ok-5 nad(p)h- nitroreductasesoongene.net/nonmun/micro40-183.pdf184 eun-mi ho et...

The Korean Journal of Microbiology, Vol. 40, No. 3, September 2004, p. 183-188Copyright�2004, The Microbiological Society of Korea

183

� �

Stenotrophomonas sp. OK-5�� NAD(P)H- Nitroreductase� ��

��1��*

��

��

TNT ��

��

Stenotrophomonas sp. OK-5��

��

��

��

NAD(P)H-nitroreductase��

��

fractions (NTRfractions I, II, III)

��

��

��

��

��

��

. ��

��

NTR fractions I, II, III��

��

��

��

��

��

��

��

. TNT��

��

��

OK-5��

NTR fractions I, II, ��

III��

��

��

��

β-mercaptoethanol��

��

��

��

��

��

��

��

��

. TNT��

��

��

��

��

OK-5��

��

NTR��

��

��

��

��

, nitrobenzene, ��

RDX��

��

��

��

��

��

2,6-DNT��

2,4-DNT��

��

��

��

��

��

. ��

OK-5��

��

NTR fraction I��

N-��

��

��

1MSDLLNADAVVQLFRTARDS20��

��

,Xanthomonas campestris

�� NTR

�� X. axonopodis

�� NTR

��

70%��

65%��

��

��

��

��

��

. ��

OK-5��

NTR fraction I��

��

��

SmOK5nrI ��

��

��

��

��

��

��

��

��

��

X. campestris��

NTR��

81%, X. axonopodis��

NTR��

75%, ��

Streptomyces avermitilis��

NTR��

30%��

��

��

��

��

, Pseudomonasputida KT2440

�� NTR (pnrB)

�� 16%

��

��

��

��

��

.

Key words��

TNT, Stenotrophomonas sp. OK-5, nitroreductase, specific activity, amino acids sequence analysis

��

� ��

� �� (1). ��

��

(group)�� hydroxylamino ��

� �� , ��

� � �� (11, 23). ��

� �� 2,4,6-trinitrotoluene (TNT)� ��

� �� , �� . TNT� �

�� (group)� ��

��

��

�� (10). �� TNT� ��

� �� (photolysis)�� (8), ��

�� TNT ��

�. �� TNT� �� nitroreductase

(NTR)� �� . TNT� ��

�� NTR nitrobenzene nitroreductase� NAD(P)H-

nitroreductase� � �� .

TNT� ��

� �� , � nitrobenzene nitroreductase� TNT

� �� 4-hydroxyl-amino-2,6-dinitrotoluene� 4-

amino-2,6-dinitrotoluene� ��, �� NAD(P)H-

nitroreductase� TNT�� 2-hydroxylamino-4,6-

dinitrotoluene� 2-amino-4,6-dinitrotoluene� ��

�� (16). TNT� ��

� �� NTR ��(cofactor)� ��

FMN� �� flavoprotein�

�� , �� (electron donor)�

� NADH� NADPH, � �� nicotinamide� ��

� �� (6).

NTR �� NADH� NADPH��

� ��

type I� type II� ��(6, 17). Type I� �� NTR

�� (oxygen-insensitive) ��

�� nitroso ��

hydroxylamine� � �� , ��

��

�� (5, 7). Type II� �� NTR ��

(oxygen-sensitive) ��, �� 7% ��

� �� . NTR

� type II� ��

�� (anion-radicals)� �� , ��

�� (reoxidation)� �� , �,

*To whom correspondence should be addressed.Tel: 041-530-1353, Fax: 041-530-1350E-mail: [email protected]

184 Eun-Mi Ho et al. Kor. J. Microbiol

�� superoxide, hydrogen

peroxide, �� hydroxyl radical ��

��(18, 22, 24). � �� TNT ��

Stenotrophomonas sp. OK-5�� NTR� ��

�� type I� ��

Enterobacter cloacae� NTR� ��

(13).

�� 1990��

�� NTR� � ��

��

�� . Salmonella typhimurium�� NTR ��

Escherichia coli� cloning� Watanabe �(25) NTR� � �

�� 1� �� , French �(9)

E. cloacae�� NTR� N-��

� �� E. coli� S.

typhimurium� NTR� 80% ��

� ��. �� Hecht �(12) NTR� �� 3� ��

�� Thermus thermophilus�� NTR� ��

� ��(nuclear magnetic resonance) ��

�� homodimer� �� monomer� � ��

domain� C-��

� ��. Kobori �(14) X-ray� ��

(crystallographic analysis)� �� E. coli� NTR� ��

�� FMN� �� homodimer��

��. Bryant �(6) E. coli� chromosomal DNA��

NTR type I� nfsA �� nfsB ��

�� cloning�� , Nokhbeh �(19)

Salmonella enterica TA1535 �� NTR� ��

snrA� � �� nfsA ��

�� . E. cloacae�� NTR �

�� Koder �(15)

pUC18 plasmid� �� subcloning�� vector�

��, E. coli�� (overexpression)��

�� pH�� NTR� �� .

�� Stenotrophomonas sp. OK-5�� NTR

�� fraction� �� 27 kDa��

(13), �� Whiteway �(26)� nitrofuran� �� E. coli��

�� 27 kDa� 24 kDa� NTR�� .

� �� TNT �� Stenotrophomonas sp. OK-5�

�� NADP(H)-NTR� ��

�� .

��

NTR� ��

NTR �� (13)� ��

�� . �� Bradford ��(4)��

�� , NTR� �� Bollag �

(3)� �� 12% SDS-PAGE �� . �

�� NTR � �� NADH� �� French

��(9)� �� . NTR� �� Tris-

HCl buffer (50 mM, pH 7.5)� �� TNT (0.1 mM), NADH

(0.2 mM)� �� NTR� �� 1 ml� ��

�� NADH� � �� 6,300

M-1�cm-1�� 340 nm�� 1� �� NADH� ��

�� . 1 unit 25oC�� 1 mole�

NADH� �� , ��(specific activity)

unit/mg�� .

NTR ��

�� 50 mM Tris-HCl

buffer (pH 7.5)� �� 1 mM� ��

�� 5�� , 0.1 mM TNT� 200 µl, 0.2 mM

NADH �� 10 µl� ��

� 1 ml� �� 1��

��. ��

100% �� .

NTR� ��

TNT� � �� NTR� ��

��. �� NTR� �� TNT� �

� �� . RDX (hexahydro-

1,3,5-trinitro-1,3,5-triazine), nitrobenzene, 2,6-DNT (2.6-dinitrotoluene),

2,4-DNT (2.4-dinitrotoluene)� �� 0.1 mM� ��

� �� 0.2 mM NADH� ��

� �� , 340 nm�� 1� �� NADH� ��

�� . TNT� ��

�� 100%� �� .

NTR� N-��

�� NTR� 12% SDS-polyacrylamide gel��

�� gel�� semidry electroblotter (Bio-Rad, Hercules, USA)

� �� PVDF membrane (Biosystems, Foster City, USA)�

� 18 V�� 20� �� . �� PVDF

membrane� coomassie blue R-250 �� 10�� ,

50%� methanol� ��

��. �� PVDF membrane� ��

�� (Model 491A, Perkin Elmer, Foster

City, USA)� �� . �� N-��

�� NCBI� BLAST search ��

�, �� .

NTR ��

�� OK-5� NTR �� PCR ��

� �� primer� ��

�� N-�� 7��

Vol. 40, No. 3 Stenotrophomonas sp. OK-5�� NADP(H)-nitroreductase� �� 185

�� emNRO101� ��, emNRO101 ��

�� 7�� nested primer�

emNRON-1� ��, �� forward primer� ��

�. �� reverse primer� �� Genbank��

� �� NTR ��

�� . � ��

� �� specific primer�

�� emNRO102� ��(Table 1). NTR ��

�� OK-5 genomic DNA� ��

� �� forward primer� reverse primer�

Premix PCR tube (AccuPower® PCR PreMix, Bioneer Co.,

Chung-Buk, Korea)� �� PCR (MJ Research,

Waltham, USA)� ��. PCR �� 94oC�� 30

�, 55oC�� 30�, 72oC�� 1� 30� �� 30 cycles �

��. DNA �� 0.8% agarose

gel� �� , gel�� DNA �

�� AccuPrep® gel purification kit (Bioneer Co., Daejon, Korea)

� �� .

NTR ��

PCR� �� OK-5 NTR �� vector

pBluescriptIIKS(+)� subcloning�� . �� vector DNA

� �� EcoRV� �� 1��

agarose �� , ��

vector� insert DNA�� 1:3 ��

�� rapid DNA ligase I (Takara Co., Seoul, Korea)�

�� 16oC�� 15~30�� . Ligation mixture�

E. coli competent cell� �� 42oC�� 30� �� heat

shock� �� , �� 100 µg/ml ampicillin� 50 µl X-

gal� �� LB �� 37oC�� 24��

�� subclone� ��. Subcloning� OK-5 NTR �

�� subclone�� DNA

� ��. NTR ��

primer� nested primer� emNRON-1� pBluescriptIIKS(+)

universal primer� M13 primer� ��

(Model 3700, ABI, Piscataway, USA)� �� .

�� OK-5 NTR �� NCBI BLAST search �

�� .

��

NTR ��

�� OK-5 NTR ��

�� , NTR fraction I� dithiothreitol, EDTA, ��

� NaCl� �� 66.7%, 43.0%, 36.8%� ��

� ��, β-mercaptoethanol ��

�� . NTR fraction II� ��

�� dithiothreitol, EDTA, �� NaCl� �� 67.3%,

41.9%, 39.4%� �� , � β-mercapto-

ethanol ��

��. NTR fraction III� dithiothreitol, EDTA, �� NaCl�

�� 59.1%, 42.1%, 39.7%� ��, β-mercapto-

ethanol� ��

(Table 1). Oh �(20)� TNT� �� P. aeruginosa��

NTR� ��

��, dithiothreitol, ascorbic acid, NaH2PO4� ��

� �� 60%�� , β-mercapto-

ethanol� Triton X-100 ��

�� .

NTR� ��

TNT� � �� NTR fractions I, II, ��

III �� . OK-5�� NTR fraction

I� TNT, nitrobenzene, �� RDX� �� 100%,

83.6%, �� 80.9%� �� 2,6-

DNT� 2,4-DNT�� 41.5%� 18.9%� ��

�. NTR fraction II �� , TNT,

nitrobenzene, RDX�� 100%, 79.9%, 76.0%� ��

��, 2,6-DNT� 2,4-DNT�� 42.3%� 17.7% ��

� ��. NTR fraction III� TNT, nitrobenzene, ��

RDX�� 100%, 81.4%, �� 79.1%� ��

�, 2,6-DNT� 2,4-DNT�� 40.7%� 18.3% ��

��(Table 2). Riefler �(21)� �� (nitro group)� ��

�� TNT ��

Table 1. Effects of specific inhibitors on the activity of nitroreductasefractions I, II, and III purified from Stenotrophomonas sp. OK-5

InhibitorsConcentration

(mM)Relative activity (%)

Fraction I Fraction II Fraction III

None 0 100 100 100

Dithiothreitol 1 66.7 67.3 59.1

EDTA 1 43.0 41.9 42.1

NaCl 1 36.8 39.4 39.7

β-mercaptoethanol 1 0 0 3

Table 2. Substrate specificities of nitroreductase fractions I, II, and IIIpurified from Stenotrophomonas sp. OK-5

SubstratesConcentration

(mM)

Relative activity (%)

Fraction I Fraction II Fraction III

TNT 0.1 100 100 100

Nitrobenzene 0.1 83.6 79.9 81.4

RDX 0.1 80.9 76.0 79.1

2,6-DNT 0.1 41.5 42.3 40.7

2,4-DNT 0.1 18.9 17.7 18.3


��, 2,6-DNT� 2,4-DNT ��

oxygenase� � ��

�� reductase� � � ��

�� . ��, � �� OK-5 NTR� ��

2,6-DNT� 2,4-DNT ��

� �� .

NTR� N-�� PCR� ��

� �� NTR� OK-5��

NTR �� NTR � ��

�� NTR fraction I N-�� .

OK-5�� NTR fraction I N-��

� �� N-�� 20

�� Met-Ser-Asp-Leu-Leu-Asn-Ala-Asp-Ala-

Val-Val-Gln-Leu-Phe-Arg-Thr-Ala-Arg-Asp-Ser� ��.

�� NCBI BLAST search ��

�� Xanthomonas campestris

NTR� X. axonopodis NTR�� 70%� 65%� ��

�� , Pseudomonas putida NTR (pnrB)

� Xylella fastidiosa NTR�� 20%� 15%�

�� (Fig. 2). Blehert �(2)� P. putida� P. fluorescens

�� NTR N-��

��, 34% �� . ��, P. putida

JLR11�� pnrA� pnrB � � NTR� �� N-��

�� 10%� � ��

� �� . � �� OK-5��

NTR N-�� P. putida pnrB� ��

�� . �� OK-5 reductase ��

� �� D/B �� nitroreductase

�� degenerate ��

�� Table 3� ��.

NTR ��

�� OK-5�� NTR fraction I� ��

SmOK5nrI� �� OK-5 genomic DNA�� SmOK5nrI �

�� , �� N-��

�� degenerate

primer� nested primer� �� PCR� ��

� �� , 770 bp �� DNA ��

��(Fig. 3). �� 770 bp DNA �� SmOK5nrI ��

Fig. 1. Sequence alignment of N-terminal seqeunce of nitroreductase fraction I with Stenotrophomonas sp. OK-5, Xanthomonas campestris,Xanthomonas axonopodis, Pseudomonas putida, Xylella fastidiosa. The nitroreductase of Stenotrophomonas sp. OK-5 showed 70% similaritywith nitroreductase of Xanthomonas campestris and 65% with nitroreductase of Xanthomonas axonopodis.

Fig. 2. PCR products of nitroreductase gene from Stenotrophomonassp. OK-5. Lanes: M, molecular size marker; 1, PCR product amplifiedwith emNRO101 and emNRO102 primers, 2, 770-bp PCR productamplified with emNRON-1 and emNRO102.

Table 3. Degenerate primers for amplification of SmOK5NRI

Oligomer name Degenerate primer sequence

emNRO101 5'-ATG ACN(A/T/G/C) GAY(C/T) Y(C/T)TA Y(C/T)TG AAN(A/T/G/C) GCT

emNRON-1 5'-TTY(C/T) M(A/C)GA ACY(C/T) GCW(A/T) CGW(A/T) GAY(C/T) CGA

emNRO102 5'-M(A/C) K(G/T)C GAW(A/T) R(A/G)CG TGC S(C/G) TTC GTCa

aRepresents sequence of the antisense strand

Vol. 40, No. 3 Stenotrophomonas sp. OK-5�� NADP(H)-nitroreductase �� 187

�� 0.8% agarose gel�� DNA ��

�� pBluescriptIIKS+ vector� subcloning��. Subcloning�

SmOK5nrI �� emNRON-1� M13

primer� ��

SmOK5nrI �� ORF� �� 631 bp ��

�� , �� NCBI

BLAST search �� ,

Xanthomonas campestris (81%), X. axonopodis (75%), Streptomyces

avermitilis (30%), P. putida KT2440 (pnrB) (16%)��

�� (Fig. 4). Koder �(15)� E. cloacae��

NTR� �� E.

coli NfsA� 87%� �� . � ��

OK-5 NTR� Xanthomonas �(genus) NTR� ��

� �� .

��, �� Stenotrophomonas sp. OK-5�� cDNA library�

�� NTR �� clone� ��, NTR

�� promoter� �� OK-5��

�� .

��

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(Received August 2, 2004/Accepted September 7, 2004)

ABSTRACT : Physiological and Molecular Characterization of NAD(P)H-Nitroreductase fromStenotrophomonas sp. OK-5 Eun-Mi Ho, Hyung-Yeel Kahng1, and Kye-Heon Oh* (Department of Life Science, Soon-chunghyang University, P.O. Box 97, Asan, Chung-Nam 336-600, Korea, 1Department of Envi-ronmental Education, Sunchon National University, Sunchon, Jeonnam 540-742, Korea)

Stenotrophomonas sp. OK-5 capable of degrading TNT has been found to have three nitroreductase fractionsdesignated as NTR fractions I, II, and III. NTR in a previous study. This study was attempted to reveal phys-iological and molecular characteristics of NTR fractions I, II, and III in strain OK-5. Several chemicals (e.g.,EDTA, NaCl, dithiothreitol, β-mercaptoethanol) were tested for their effect on enzyme activity of NTRs, dem-onstrating that enzyme activities of NTR fractions I, II, and III from OK-5 were inhibited in the presence of β-mercaptoethanol. Substrate specificity test showed that NTR fractions I, II, and III all have over 70% enzymeactivities for nitrobenzene or RDX as a substrate. N-terminal amino acid sequence of NTR fraction I fromStenotrophomonas sp. OK-5 was 1MSDLLNADAVVQLFRTARDS20 and exhibited 70% sequence homologywith that of NTR from Xanthomonas campestris. NTR I gene from Stenotrophomonas sp. OK-5 (SmOK5nrI)shared extensive sequence homology in deduced amino acid sequence of PCR product with NTRs from Xan-thomonas campestris (81%), X. axonopodis (75%), Streptomyces avermitilis (30%), whereas they had lowhomology with that from P. putida KT2440 (pnrB) (16%).

stenotrophomonas sp. ok-5 nad(p)h- nitroreductasesoongene.net/nonmun/micro40-183.pdf184 eun-mi ho et...

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