2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its SubstrateSpecificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid underOxidizing Conditions

Yong-Hak Kim,a Woo-Seok Song,b Hayoung Go,b Chang-Jun Cha,c Cheolju Lee,d Myeong-Hee Yu,d Peter C. K. Lau,e Kangseok Leeb

Department of Microbiology, Catholic University of Daegu School of Medicine, Nam-Gu, Daegu, Republic of Koreaa; Department of Life Science, Chung-Ang University,Seoul, Republic of Koreab; Department of Biotechnology (BK21 Program), Chung-Ang University, Anseong, Republic of Koreac; BRI, Korea Institute of Science andTechnology, Seongbuk-Gu, Seoul, Republic of Koread; Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada, Departments ofChemistry and Microbiology & Immunology, McGill University, Montreal, Quebec, Canada, and FRONT Centre in Green Chemistry and Catalysis, Montreal, Quebec,Canadae

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitroben-zoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison revealsthat NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216.The truncated mutant �65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaf-fected; however, both activities were abolished in the �193-216 mutant, suggesting that these regions are necessary for the catal-ysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and thereduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicatingoxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5=-dithio-bis-(2-nitrobenzoicacid) and ZnCl2, which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H2O2. SDS-PAGE and mass spectrometry revealed the for-mation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagene-sis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificitytoward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential spec-ificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizingconditions.

Nitroreduction, catalyzed by an NAD(P)H-dependent nitrore-ductase, is the essential first step in the catabolism of a variety

of structurally diverse nitroaromatic compounds, such as nitro-benzoates, nitrotoluenes, and 3-nitrophenols. Biodegradation of2-nitrobenzoic acid (2-NBA) by Pseudomonas fluorescens strainKU-7 is the first prokaryotic example of formation of a 3-hydroxy-anthranilate intermediate (1, 2). Iwaki and colleagues (3) identi-fied the nbaA and nbaB genes, which encode 2-nitroreductase(NbaA) and a mutase (NbaB) that mediate NADPH-dependentreduction of 2-NBA to 2-hydroxylaminobenzoic acid and a Bam-berger-type rearrangement of 2-hydroxylaminobenzoic acid to3-hydroxyanthranilate. In Arthrobacter protophormiae strainRKJ100, there is an alternative route of 2-NBA metabolism toform 3-hydroxyanthranilate and anthranilate (4, 5).

NbaA (GenBank accession number BAF56676.1) is a homodi-meric NADH:flavin mononucleotide (FMN) oxidoreductase-likefold protein (3). It is similar to a putative flavin-containing pro-tein (78% sequence identity; ABE46991.1) located on the Polaro-monas sp. strain JS666 plasmid 1 (GI:91790731), and it includes aflavin reductase-like domain (Pfam accession number PF01613 inthe Pfam database [http://www.sanger.ac.uk/Software/Pfam/])(6). Structurally, it is related to the NADH:FMN oxidoreductase-like structural family (SCOP accession number b.45.1.2 or 50482;http://scop.berkeley.edu/) (7). Iwaki and colleagues (3) showedthat Asn40, Asp76, and Glu113 in the conserved region of NbaAare necessary for binding to a divalent metal ion implicated inFMN binding, and that an insertion loop of 10 amino acids at

positions 65 to 74 mediates NADPH binding. It exhibits a narrowspecificity toward 2-NBA, which is different from other nitro/flavin reductases (8–10). However, the catalytic properties andsubstrate specificity of NbaA have not been characterized.

The aim of this study was to determine the catalytic propertiesand substrate specificity of NbaA. Recently, it was found thatNbaA acts as a redox-sensitive protein which is able to form high-er-order disulfide-bonded proteins under oxidizing conditions(11). We observed that the enzyme was able to reduce 2-NBA and2,4-dinitrobenzoic acid (2,4-DNBA) at different lag times, andthat those activities were gradually decreased during the forma-tion of intermolecular disulfide bonds under aerobic conditions.The formation of intermolecular disulfide bonds was analyzed bySDS-PAGE and tandem mass spectrometry. In order to confirmthe reduction of 2-NBA and 2,4-DNBA, the products derivatizedwith acetic anhydride were analyzed by thin-layer chromatogra-phy and high-performance liquid chromatography with tandem

Received 22 October 2012 Accepted 23 October 2012

Published ahead of print 2 November 2012

Address correspondence to Yong-Hak Kim, ykim@cu.ac.kr, or Kangseok Lee,kangseok@cau.ac.kr.

Y.-H.K., P.C.K.L., and K.L. are co-senior authors.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.02016-12

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mass spectrometry. We performed deletion mutations in the vari-able regions (�65-74 and �193-216) to examine the role of thoseregions in the catalysis and specificity of NbaA. Site-directed mu-tagenesis was carried out to examine the possible roles of cysteinesat positions 39, 103, 141, and 194 in oxidative modification of theenzyme.

MATERIALS AND METHODSMaterials and chemicals. The following reagents were purchased fromSigma (St. Louis, MO): 5,5=-dithio-bis-(2-nitrobenzoic acid) (DTNB),dithiothreitol (DTT), 2,4- and 3,5-dinitrobenzoic acids (DNBA), EDTAdisodium salt, N-ethylmaleimide (NEM), hydrogen peroxide (H2O2),isopropyl ß-D-thiogalactopyranoside (IPTG), ß-NAD 2=-phosphate, re-duced form (NADPH), 2-, 3- and 4-nitrobenzoic acids (NBA), and ribo-flavin 5=-monophosphate (FMN) sodium salt dehydrate. Sequencing-grade porcine trypsin was obtained from Promega (Madison, WI). A fastprotein liquid chromatography system, columns, and resins were sup-plied by GE Healthcare Life Sciences (Uppsala, Sweden). Protein mini-gelkits and assay reagents were supplied by Bio-Rad (Hercules, CA) andPierce (Thermo Fisher Scientific Inc., Rockford, IL). All other solventsand reagents used were of analytical grade and were obtained from USBCorporation (Cleveland, OH), J. T. Baker (Phillipsburg, NJ), and Merck(Darmstadt, Germany).

Strains and culture conditions. The complete DNA sequence ofNbaA was PCR amplified and cloned into pSD80 as previously described(3). The truncated (�65-74 and �193-216) and site-specific mutants(C39A, C103A, C141A, C194A, and C141A C194A) were constructed us-ing the PCR primers given in Table 1. The resulting plasmids were trans-formed into Escherichia coli strain BL21(DE3). Transformed E. coli strainswere grown in LB broth at 37°C and 180 rpm in the presence of 100 mgliter�1 ampicillin. When cells reached an optical density at 600 nm(OD600) of �0.5, the recombinant proteins were induced with 0.2 mMIPTG for 1 h. An empty pSD80 plasmid-transformed strain was includedunder the same culture conditions as a control.

Protein purification. Cells were harvested at 4°C at 3,000 � g for 15min using a Sorval centrifuge and rotor. Washed cells were suspended in3 volumes of buffer A containing 10 mM DTT, 1 mM EDTA, and 50 mM

Tris-HCl (pH 7.4) and disrupted by 3 passages through a prechilledFrench pressure cell (maximum capacity, 3.5 ml). After centrifugation at12,000 � g for 30 min, the supernatant was applied to a DEAE-Sepharosecolumn (1.6 by 20 cm) equilibrated with buffer A at a flow rate of 2 mlmin�1, and bound proteins were eluted by a 40-min linear gradient to 0.6M NaCl plus buffer A with collection of 2-ml fractions. Aliquots of eachfraction were mixed into 50 mM sodium phosphate buffer (pH 7.4) con-taining 1 mM 2-NBA, 1 mM NADPH, 10 �M FMN, and 0.1 mM MnCl2to determine the rate of NADPH oxidation at 340 nm (ε340 � 6.21 mM�1

cm�1) using a Shimazu UV-1800 spectrophotometer (Shimazu Co.,Kyoto, Japan) at room temperature. Fractions showing more than half-maximum activity were combined and treated with 1 M ammonium sul-fate prior to loading on a Phenyl-Sepharose column (1.6 by 10 cm) equil-ibrated with 1 M ammonium sulfate in buffer A at 1 ml min�1. Boundproteins were eluted in a 40-min linear gradient to buffer A with collectionof 1-ml fractions. Fractions containing more than half-maximum activitywere combined as described above and concentrated to approximately500 �l using Centriplus YM-30 centrifugal filter devices (Millipore Co.,Bedford, MA) before application to a Superdex 200 column (1.6 by 60 cm)equilibrated with buffer A at a flow rate of 0.25 ml min�1. Protein con-centration was determined using a Pierce Coomassie Plus protein assay kitand bovine serum albumin as a standard.

Enzyme reconstitution. Purified NbaA (final concentration, 0.1 �Mmonomer) was mixed with various concentrations of divalent metal chlo-ride salts (CaCl2, MgCl2, and MnCl2) in 50 mM sodium phosphate buffer(pH 7.4) containing 1 mM NADPH, 10 �M FMN, 1 mM 2-NBA, or2,4-DNBA, and NADPH oxidation was monitored for 30 min at roomtemperature. The lag time for enzyme activity to 2-NBA or 2,4-DNBA wasestimated by a logistic function, and the metal-binding affinity (K0.5M)was estimated by the Hill equation as mentioned below. The enzymeturnover number (kcat, s�1) was calculated by dividing the maximumvelocity, vmax, by the 0.05 �M concentration of NbaA homodimer as thebasic catalyst.

Enzyme inhibition. To examine the presence of reactive thiol/thiolatein NbaA, 0.1 �M NbaA monomer was treated with various concentra-tions of a sulfhydryl-blocking agent (DTNB) or a sulfhydryl complexingagent (ZnCl2) in 50 mM sodium phosphate buffer (pH 7.4) containing 10

TABLE 1 PCR primers used for construction of wild-type and mutant NbaA

PCR primer Nucleotide sequence Description Reference or source

pSD80F 5=-GAGCTGTTGACAATTAAT-3= pSD80 vector primers for DNA sequencing 45pSD80R 5=-AGGACGGGTCACACGCGC-3=NbaA-F 5=-CGGAATTCATGACGCACATTGCAATGTCA-3= PCR amplification of a full DNA sequence containing

the nbaA gene3

NbaA-R 5=-AAAACTGCAGTCAGGGAGTAATCGGAAAGA-3=NbaA-64R-blunt 5=-ATAATGATCCACGGCGAT-3= Construction of �65-74 mutant by blunt ligation of

PCR productsThis study

NbaA-75F-blunt 5=-AAAGACACGCTAAAA AACATC-3=NbaA-R192 5=-GTCGGTGCTGCAGCATTAGTTGGGTCCTCC-3= Construction of �193-216 mutant This studyNbaA-C39A-R 5=-AAGCGCTATAAGGCGCGGCATTCGCCAACCC

CTCACTGTTCAAACTT-3=Site-directed mutagenesis of cysteine at position 39

into alanine (C39A)This study

NbaA-139F 5=-TGCCGCGCCTTATAGCGCTT-3=NbaA-399R 5=- AAACCATTCGCTCAGCAATA-3= Site-directed mutagenesis of cysteine at position 103

into alanine (C103A)This study

NbaA-C103A-F 5=-TATTGCTGAGCGAATGGTTTTGGCGGGTAGTGATTTCCCCTCTCATAT-3=

NbaA-C141A-R 5=-AATCGATGATTTTGTAGAGTTTCGCTTCCCACGCAATGGGTGCGTCGG-3=

Site-directed mutagenesis of cysteine at position 141into alanine (C141A)

This study

NbaA-445F 5=-ACTCTACAAAATCATCGATT-3=NbaA-557R 5=-AGTTGGGTCCTCCCAAGCGCC-3= Site-directed mutagenesis of cysteine at position 194

into alanine (C194A or C141A/C194A)This study

NbaA-C194A-F 5=-GGCGCTTGGGAGGACCCAACTATGCGCGAACCACCGACCGGGTTCGCC-3=

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�M FMN and 0.1 mM MnCl2 for 10 min before enzyme activity wasmeasured by addition of 1 mM NADPH and 1 mM 2-NBA.

Determination of substrate specificity and optimal conditions. Sub-strate specificities of wild-type and mutant NbaA proteins were analyzedwith various concentrations of 2-/3-/4-NBA and 2,4-/3,5-DNBA in 50mM Tris-HCl (pH 7.4) containing 1 mM NADPH, 10 �M FMN, and 0.1mM MnCl2. The lag time for equilibration of enzyme activity with a sub-strate was estimated by a logistic function fit on the continuous NADPHoxidation (�A340) curve, which was monitored every 2 s for 30 min. Amidpoint rate of NADPH oxidation (v) by substrate concentration[S] was used for curve fitting with the Hill equation: v � (vmax � [S]h)/(K0.5Sh � [S]h), in which vmax, K0.5S, and h are the apparent maximumvelocity, half-saturation constant, and Hill coefficient for substrate bind-ing, respectively. Temperature and pH optima of NbaA reacting with2-NBA were determined by varying the temperature in 50 mM sodiumphosphate buffer at pH 7.4 or by varying the buffer pH at 25°C. In order toexamine substrate-induced effects on the enzyme activity and differentialspecificity for 2-NBA and 2,4-DNBA, reconstituted NbaA at a final con-centration of 0.1 �M monomer in 50 mM sodium phosphate buffer (pH7.4) containing 0.1 mM MnCl2 and 10 �M FMN was preincubated atambient conditions for 30 min with 0.5 mM either 2-NBA or 2,4-DNBAor with 1 mM NADPH. The enzyme reaction was then started with theremaining substrate.

Analysis of metabolites. Metabolites derived from the reduction of2-NBA and 2,4-DNBA by NbaA were analyzed by thin-layer chromatog-raphy (TLC) and high-performance liquid chromatography with tandemmass spectrometry (HPLC-MS/MS) after following a modified procedureof derivatization of the unstable hydroxylamine groups with acetic anhy-dride (12). Reconstituted NbaA at a final concentration of 0.1 �M mono-mer was added to 10 ml of 50 mM sodium phosphate buffer (pH 7.4)containing 0.5 mM either of 2-NBA or 2,4-DNBA, 0.1 mM MnCl2, and 10�M FMN. After mixing with or without 1 mM NADPH, the reactionswere carried out under aerobic or anaerobic conditions with monitoringNADPH oxidation for 50 min at room temperature. In order to removedissolved oxygen, all buffers and solutions used were treated with sodiumsulfite prior to mixing the enzyme and reactants. Aerobic or anaerobicconditions of the reaction mixtures were demonstrated by adding a redoxindicator, leucomethylene blue for the so-called blue bottle reaction (13).The enzyme reaction was stopped by cooling in an ice bath, mixed with 2.5ml of methanol, and acidified by slowly adding 7.5 ml of HCl. When aceticanhydride (7.5 ml) was added dropwise, the temperature was maintainedat 0°C. After 30 min, the derivatization was stopped by the addition ofsodium acetate (8.75 mg) dissolved in 15 ml of water. After repeatedextraction with ethyl acetate, the combined organic layers were washedwith water and dried over MgSO4. The solvent was evaporated, and equalamounts (10 �l) of metabolites dissolved in 1 ml methanol were chro-matographed on silica gel for separation with 2:2:1 (vol/vol/vol) hexane–t-butyl methyl ether-ethyl acetate. For HPLC-MS/MS analysis, a ThermoAccela UHPLC system was connected with a reverse-phase Hypersil Goldcolumn (100 by 2.1 mm; particle size, 1.9 �m) to an LTQ-Velos Massinstrument. The column was equilibrated with 95% buffer A (0.1% for-mic acid in H2O) plus 5% buffer B (0.1% formic acid in acetonitrile) at aflow rate of 150 �l min�1. After injection of 10 �l sample prepared by10-fold dilution of the concentrated metabolites in methanol, metaboliteswere eluted with a linear gradient of 5 to 80% buffer B over 30 min. Thespray voltage was set with 5-kV negative polarity, and the temperature ofthe heated capillary was set to 275°C. Survey full-scan MS spectra (m/z 50to 500) were acquired with 1 microscan and a resolution of 10,000 fordetermination of precursor ions and charge states, and MS/MS spectra ofthe 3 most intense ions from the survey scan were acquired with thefollowing options: isolation width, �1 Da; normalized collision energy,35%; dynamic exclusion duration, 20 s. The mass spectral data were ana-lyzed with Thermo Xcalibur software v. 2.1.

Analysis of NbaA oligomerization. Oligomerization of NbaA was an-alyzed by size-exclusion chromatography at 0, 2, 6, and 12 h after incuba-

tion of PD10-desalted NbaA in 50 mM Tris-HCl buffer (pH 7.4) at anambient temperature. Protein elution from a Superdex 200 column (1.6by 60 cm) equilibrated with 50 mM Tris-HCl buffer (pH 7.4) at a constantflow rate of 0.25 ml min�1 was monitored at 280 nm with collection of0.5-ml fractions. In each fraction, the composition of oligomeric speciesof native protein was determined by native PAGE. The intensity of Coo-massie-stained protein was calculated by local average volume using Mo-lecular Dynamics ImageQuant, version 5.2 (GE Healthcare Life Sciences,Piscataway, NJ).

Determination of physicochemical factors for disulfide bond for-mation. To examine physicochemical factors affecting disulfide bondformation, wild-type NbaA protein was incubated at a 1 or 10 �M con-centration in various buffers (50 mM morpholineethanesulfonic acid-NaOH, pH 5.5 to 6.5; 50 mM HEPES-HCl, pH 6.5 to 8.1; 50 mM Tris-HCl, pH 7.8 to 9.7) containing DTT (0 to 10 mM), H2O2 (0 to 10 mM), orNaCl (0 to 1 M). During incubation at 25 or 37°C, subsamples werecollected and treated with 10 mM NEM for 1 h in darkness. The compo-sitions of the disulfide-bonded proteins were analyzed by nonreducingSDS-PAGE.

Mass spectrometry. Disulfide-bonded isomer bands of NEM-treatedproteins were excised from a gel and digested with trypsin for mass spec-trometry (14). Dried peptide extracts were dissolved in 0.4% acetic acidand analyzed with a nanoflow LC-LTQ linear ion trap mass spectrometer(Thermo Fisher Scientific Inc.) with an Agilent Series 1200 nanoflow liq-uid chromatography system (Agilent, Santa Clara, CA) and a capillarycolumn (75-�m inner diameter, 360-�m outer diameter, 15-cm length),which was packed in house with Magic C18AQ particles (5 �m; 200-Åpore size; Michrom Bioresources, Inc., Auburn, CA). The chromato-graphic conditions comprised a 45-min linear gradient from 5 to 40%acetonitrile (ACN) in 0.1% formic acid (FA), followed by a 5-min columnwash in 80% CAN– 0.1% FA and a 10-min column re-equilibration with5% CAN– 0.1% FA at a flow rate of 0.35 �l min�1. The full mass scan wasperformed between m/z 300 and 2,000 and was followed by 5 data-depen-dent MS/MS scans with the following options: isolation width, �1.5 m/z;normalized collision energy, 25%; dynamic exclusion duration, 30 s. Massdata were analyzed by an in-house Excel macro program with the optionsof average mass (m/z); precursor ion mass tolerance, 0.8 Da; fragmentmass tolerance, 1 Da; and 2H loss (�2.02 Da) from all combinations of 2tryptic peptides, each containing cysteine.

Western blotting. Protein concentrations of wild-type and mutantNbaA proteins in whole-cell extracts were determined using a rabbit poly-clonal anti-NbaA antibody and purified NbaA standard. Standard ECLreagents and films (GE Healthcare Life Sciences, Piscataway, NJ) wereused for Western blot detection with horseradish peroxidase (HRP)-con-jugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, Inc., SantaCruz, CA).

RESULTSComparative sequence analysis of NbaA with flavin-containingreductase-like proteins. In order to infer the enzymatic proper-ties of NbaA, the amino acid sequence was reexamined by analyz-ing its phylogenetic relationship to the currently availableGenBank protein database. The NbaA sequence (216 amino acids)is most similar to a putative flavin-containing reductase-like(FlaRed) protein (78% sequence identity; protein accession num-ber ABE46991.1) retrieved from Polaromonas sp. strain JS666plasmid 1 (GI:91700611:235953-236603). A phylogenetic tree ofNbaA and homologous FlaRed proteins indicates its narrow oc-currence among soil- and plant-associated species of alpha- andbetaproteobacteria (Fig. 1A). Several strains, including Polaromo-nas sp. strain JS666, contain two or more homologous FlaRedprotein-encoding genes (match scores of 100 and E valuesof 10�30), and some bacteria carry FlaRed-coding plasmids. Thesequence of NbaA shows 32% identity to the structurally charac-

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terized MTH152 (Protein Data Bank [PDB] accession number1EJE) of Methanobacterium thermoautotrophicum strain �H (15).Sequence alignment shows a conserved cysteine (C141) and twovariable regions, V1 and V2, corresponding to amino acids 65 to74 and 193 to 216 in NbaA (Fig. 1B). In the NbaA polypeptide,there are 4 cysteines at positions 39, 103, 141, and 194.

Substrate specificities of NbaA and its mutants deleted forthe variable regions. To examine potential roles of the variableregions (V1 and V2), wild-type NbaA and truncated mutant pro-teins �65-74 and �193-216 were overproduced in E. coli cells andpurified up to nearly 95% homogeneity as seen by SDS-PAGE(Fig. 2A). In size-exclusion gel chromatography, the elution peaks

of NbaA (24.4 kDa) and the truncated mutant proteins, �65-74(23.3 kDa) and �193-216 (21.7 kDa), were detected in the molec-ular mass range of 40 to 50 kDa, which corresponds to their pre-dicted homodimer mass values.

Figure 2B and C show the optimal pH and temperature for invitro 2-NBA reduction activity. The relative activity (%) of NbaAwas optimal at pH 7.4 and 50°C. However, its activity sharplydeclined above pH 7.4 and 50°C. In this study, we determined theenzyme properties and substrate specificities of wild-type and mu-tant NbaA in 50 mM sodium phosphate buffer, pH 7.4, at 25°C.Under this condition, NbaA was able to reduce 2-NBA and 2,4-DNBA but not 3-NBA, 4-NBA, or 3,5-DNBA. The �65-74 mutant

FIG 1 (A) Phylogenetic tree of Pseudomonas fluorescens KU-7 NbaA (GenBank accession number BAF56676.1; shown in the shaded box) and homologousflavin-containing reductase [FlaRed]-like proteins clustered by the unweighted-pair group method using average linkages (UPGMA). The scale bar indicates 0.1amino acid changes per branch length. (B) Partial sequence alignment of NbaA (BAF56676.1:KU-7) and representative homologues highlights the conservedCys141 site and the variable V1 (amino acids 65 to 74) and V2 (193 to 216) regions. Two or more copies of homologous genes are present in several bacterialspecies (GenBank accession numbers): Azorhizobium caulinodans ORS571 (BAF88487.1 and BAF88982.1), Agrobacterium radiobacter K84 (ACM26428.1 andACM26650.1), Bradyrhizobium japonicum USDA 110 (BAC48596.1, BAC48663.1, and BAC47690.1), Bradyrhizobium sp. strain BTAi1 (ABQ35041.1 andABQ34396.1), Bradyrhizobium sp. strain ORS278 (CAL76387.1 and CAL75771.1), Burkholderia phytofirmans PsJN (ACD19612.1 and ACD21190.1), Burkhold-eria xenovorans LB400 (ABE34598.1 and ABE33052.1), Polaromonas sp. strain JS666 (ABE46991.1 [plasmid 1], ABE43004.1, and ABE45686.1), Pseudomonasfluorescens Pf0-1 (ABA74886.1 and ABA76199.1), Rhizobium etli CFN 42 (ABC91672.1 and ABC90974.1), Rhizobium etli CIAT 652 (ACE92008.1 andACE91236.1), Rhizobium leguminosarum bv. trifolii WSM1325 (ACS57175.1 and ACS56319.1), Rhizobium leguminosarum bv. trifolii WSM2304 (ACI55925.1and ACI55127.1), and Rhizobium leguminosarum bv. viciae 3841 (CAK08853.1, CAK06476.1, and CAK08013.1). FlaRed-coding plasmids were Agrobacteriumvitis S4 pAtS4e (ACM40198.1), Burkholderia phymatum STM815 pBPHY01 (ACC74970.1), Polaromonas sp. strain JS666 plasmid 1 (ABE46991.1), Sinorhizobiummeliloti 1021 pSymA (AAK65563.1), and Sinorhizobium medicae WSM419 pSMED02 (ABR64490.1).

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had 2-NBA activity similar to that of wild-type NbaA; however,the mutant displayed approximately 20-fold lower 2,4-DNBA ac-tivity than wild-type NbaA, whereas the �193-216 mutant hadapproximately 9-fold lower 2-NBA activity and no 2,4-DNBA ac-tivity (Fig. 2D). These results indicate that the V1 region is neces-sary for 2,4-DNBA but not 2-NBA, while the V2 region affects thecatalysis of both 2-NBA and 2,4-DNBA.

Effects of divalent cations on NbaA reduction of 2-NBA and2,4-DNBA. Purified NbaA without addition of a divalent cationshowed a significant delay (�6 min) for the 2-NBA activity and no2,4-DNBA activity for 30 min (Fig. 3A and B). Treatments ofpurified NbaA with chloride salts of divalent cations, includingCaCl2, MgCl2, and MnCl2, markedly enhanced the enzyme activ-ity for both substrates by decreasing the lag times. Ca2� and Mn2�

ions produced similar activities for 2-NBA; however, the metal ionbinding affinities and lag times of the enzyme were significantlydifferent from each other (Table 2). The Mn2� ion had a higherbinding affinity (K0.5M � 25 �M) than the Ca2� ion (K0.5M � 330�M), while the Mn2�-bound enzyme displayed approximately3-times-lower activity and a longer lag time for 2,4-DNBA reduc-

tion than the Ca2�-bound enzyme. In contrast, NbaA had similaraffinities for binding Mg2� and Ca2� ions, but the Mg2� ion con-ferred much lower enzyme activities and longer lag times for bothsubstrates than Mn2� and Ca2� ions. Furthermore, the Mg2�-bound enzyme was rapidly decreased (inactivated) after equilibra-tion. The binding affinities of each cation to purified NbaA react-ing with 2-NBA and 2,4-DNBA did not significantly differ,indicating that the divalent cations did not affect substrate speci-ficity of NbaA to 2-NBA and 2,4-DNBA.

It is worth noting that the reduction of 2-NBA and 2,4-DNBA by NbaA does not occur simultaneously: the reductionof 2-NBA occurs earlier than that of 2,4-DNBA. Moreover, thereduction of 2,4-DNBA, but not 2-NBA, failed in the presenceof 1 mM DTT. Figure 3C shows that NADPH turnover rates(kcat) of NbaA for 2-NBA and 2,4-DNBA are directly propor-tional to the first-order loss of enzyme activity, which is corre-lated with the type of metal ions. When assayed with 2-NBA,the enzyme was inhibited by a thiol-blocking agent, DTNB, anda thiolate-complexing agent, ZnCl2, with 50% inhibitory con-centrations (IC50s) of 19 and 330 �M (Fig. 3D). This inhibition

FIG 2 (A) Overexpression and purification of wild-type NbaA and the truncated mutant proteins �65-74 and �193-216. Lane 1, whole-cell extracts; 2,DEAE-Sepharose; 3, Phenyl-Sepharose; 4, Superdex 200 gel filtration. Mr, Bio-Rad Precision Plus molecular weight protein standards, in thousands (Lot161-0375). (B and C) pH (B) and temperature optima (C) of NbaA for 2-NBA. Rates of NADPH oxidation at 340 nm were measured with 50 mM sodiumphosphate buffers containing 0.1 �M NbaA monomer, 1 mM NADPH, 1 mM 2-NBA, 0.1 mM MnCl2, and 10 �M FMN. (D) NADPH oxidation rates ofwild-type NbaA (circles) and truncated mutants �65-74 (triangles) and �193-216 (squares) for the reduction of 2-NBA (open symbols) and 2,4-DNBA (closedsymbols) in 50 mM sodium phosphate buffer (pH 7.4) at 25°C. Curves were fitted by the Hill equation as described in Materials and Methods.

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was not eliminated after PD10 desalting, indicating the irre-versible inhibition of reactive thiol/thiolate groups of cysteineresidues. It was suspicious that any modifications of cysteineresidues on NbaA might induce conformational changes of theenzyme, leading to alterations in catalysis and specificity.

Effects of oxygen on metabolism of 2-NBA and 2,4-DNBA byNbaA. When NbaA was preincubated with 0.5 mM either of

2-NBA or 2,4-DNBA or with 1 mM NADPH under aerobic con-ditions, resulting in blue color after the addition of a redox indi-cator of leucomethylene blue, the enzyme activities for the twosubstrates, 2-NBA and 2,4-DNBA, were significantly decreased(Fig. 4A). After 30 min of preincubation of reconstituted NbaAwith any substrate, the addition of the remaining substrates re-sulted in approximately 30% decreased NADPH oxidation for2-NBA and about 75% decreased activity for 2,4-DNBA com-pared to results without preincubation. Under anaerobic condi-tions maintained by reducing dissolved oxygen with sodium sul-fite, NbaA showed NADPH oxidation only for 2-NBA, not for2,4-DNBA. These results indicated that NbaA was sensitive to ox-ygen, by which the enzyme activity for 2-NBA and 2,4-DNBA wasmodified and further inactivated over time during the incubationunder aerobic conditions.

In order to examine the reduction of 2-NBA and 2,4-DNBA byanalytical TLC and HPLC-MS/MS, it was necessary to derivatizeunstable metabolites of 2-NBA and 2,4-DNBA with acetic anhy-dride. The derivatization of 2-NBA metabolites allowed us to de-tect a fluorescent product band on silica gel under UV light(Fig. 4B). This product was produced from the reduction of2-NBA under anaerobic as well as aerobic conditions in the pres-

FIG 3 Continuous spectrophotometric assays for NADPH-dependent NbaA reactions for the reduction of 2-NBA (A) and 2,4-DNBA (B). Absorbance at 340 nmwas recorded after 0.1 �M NbaA monomer was immediately mixed for �3 s into 50 mM sodium phosphate buffer (pH 7.4) containing 10 �M FMN, 1 mMNADPH, and 1 mM 2-NBA or 2,4-DNBA in the presence of a metal chloride: 1 mM CaCl2, 1 mM MgCl2, and 0.1 mM MnCl2. Untreated (no addition) andMnCl2-treated NbaA were used to show effects of divalent cations and a reducing agent (1 mM DTT) on NbaA activities. (C) First-order relationship of NADPHturnover rates and levels with lag times for equilibration of metal-bound NbaA with 2-NBA and 2,4-DNBA. (D) Logistic curves for determination of half-maximal inhibitory concentrations (IC50) of a thiol-blocking agent, 5,5=-dithio-bis-(2-nitrobenzoic acid) (DTNB), and a thiolate-complexing agent, ZnCl2.Inhibitors were treated with 0.1 �M NbaA monomer in 50 mM sodium phosphate buffer (pH 7.4) containing 10 �M FMN and 0.1 mM MnCl2 for 10 min beforethe enzyme activity was measured with 1 mM NADPH and 1 mM 2-NBA.

TABLE 2 Turnover rates and metal binding affinities of NbaA

Substrate

Parametera result for:

2-NBA 2,4-DNBA

kcat (s�1) K0.5M (�M) kcat (s�1) K0.5M (�M)

No addition 12 � 1 NA ND NACaCl2 138 � 37 330 � 45 5.3 � 0.3 310 � 55MgCl2 25 � 4 290 � 43 1.1 � 0.6 280 � 58MnCl2 145 � 26 25 � 6 1.7 � 0.3 28 � 6a Parameters estimated by the Hill equation fitted to the plot of the NADPH oxidationrate by metal ion concentration with the addition of 0.1 �M NbaA monomer into 50mM sodium phosphate buffer (pH 7.4) containing 1 mM NADPH, 10 �M FMN, and 1mM 2-NBA or 2,4-DNBA at 25°C are shown as the means � standard deviations (SD).NA, not available; ND, not detected after 30 min.

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ence of NADPH but not without NADPH, suggesting that NbaAcould catalyze the reduction of 2-NBA with the consumption ofNADPH in anaerobic conditions. From TLC, 2,4-DNBA levelsappeared to be decreased by the NADPH consumption of NbaAunder aerobic conditions but not in anaerobic conditions. It was

similar to results as measured by a spectrophotometer. However,the reduction of 2,4-DNBA with NADPH under aerobic condi-tions did not show any fluorescent or visible product band onsilica gel under UV light. Thus, it was uncertain whether 2,4-DNBA actually had been reduced.

FIG 4 (A) Continuous spectrophotometric assays for the reduction of 2-NBA and 2,4-DNBA before or after 30 min of preincubation of 0.1 �M NbaA monomerwith 1 mM NADPH or 0.5 mM either of 2-NBA or 2,4-DNBA in 50 mM sodium phosphate buffer (pH 7.4) containing 10 �M FMN and 0.1 mM MnCl2 underaerobic (oxic) or anaerobic (anoxic) conditions and treated with Na2SO3. After a 50-min reaction started by the addition of the remaining substrates, aerobic oranaerobic conditions were indicated by adding a redox indicator, leucomethylene blue for the blue bottle reaction (13), as shown in the middle of the graphs. (B)Silica gel thin-layer chromatography of 2-NBA and 2,4-DNBA metabolites shows the reduction of substrate levels and the resulting product bands under UVlight. Metabolites resulting from NbaA reactions with 0.5 mM 2-NBA or 2,4-DNBA in the presence or absence of NADPH under aerobic or anaerobic conditionswere derivatized with acetic anhydride, as described in Materials and Methods. (C) Base peak chromatograms of 2-NBA metabolites resulting from NbaAreactions with 0.5 mM 2-NBA in the presence (gray peaks) or absence (dark peaks) of 1 mM NADPH under aerobic conditions. The lower panels show tandemmass spectra of m/z 166.10 and m/z 178.22 ions assigned to the fragments of 2-NBA and N-acetylanthranilic acid (product I). (D) Base peak chromatograms of2,4-DNBA metabolites resulting from NbaA reactions with 0.5 mM 2,4-DNBA in the presence (gray peaks) or absence (dark peaks) of 1 mM NADPH underaerobic conditions. The lower panels show tandem mass spectra of m/z 211.07 and m/z 239.11 ions assigned to the fragments of 2,4-DNBA and 2-acetoxyamino-4-nitrobenzoic acid (product II).

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To address this question, a sensitive HPLC-MS/MS instrumentwas employed to identify metabolites resulting from the reductionof 2-NBA and 2,4-DNBA with the consumption of NADPH byNbaA under aerobic conditions. From base peak chromatograms,the peaks of 2-NBA substrate were detected at m/z 166.11 in theretention time (RT) range of 12.29 to 12.66 min (Fig. 4C). Fromthe reaction with NADPH for 50 min, the 2-NBA level was de-creased by about 81% compared to that without NADPH. A tan-dem mass spectrum of 2-NBA contained a major fragment ionpeak at m/z 122.14 which was assigned to the fragment from col-lision-induced dissociation (CID) of carboxyl anion from the mo-lecular ion. When 2-NBA was reduced, a single peak of product Iwas detected at m/z 178.22 (RT, 15.69 min). This product gener-ated 3 major fragment ions at m/z 134.13 [M-COO�], 135.13[M-CH3CHO], and 136.01 [M-CH3CO] by collision-inducedfragmentation. Based on this, it was identified as N-acetylanthra-nilic acid (CAS no. 89-52-1), which is likely formed from the de-rivatization of 2-hydroxylaminobenzoic acid with acetic anhy-dride. The emission of a blue fluorescence on silica gel supportsthe identification of product I as N-acetylanthranilic acid. Duringthe same reaction time, the 2,4-DNBA level detected at m/z 211.07and 18.73 min was decreased with NADPH by a third of the con-trol level without NADPH (Fig. 4D). The resulting product II wasdetected at m/z 239.11 and 25.36 min. A tandem mass spectrum of2,4-DNBA had a major fragment ion peak at m/z 167.10, whichwas assigned to the loss of carboxyl anion from the molecular ion.

From CID fragmentation, product II generated a major fragmention at m/z 195.06, which was assigned to the loss of carboxyl anionfrom the molecular ion (m/z 239.11). The product II at m/z 239.11was identified as 2-(acetoxyamino)-4-nitrobenzoic acid that waslikely formed from the derivatization of 2-(hydroxylamino)-4-nitrobenzoic acid with acetic anhydride. The 2-hydroxylaminemetabolites most likely can be produced from the 2-nitroreduc-tion of 2-NBA and 2,4-DNBA by NbaA as a specific enzyme gen-erally shows excellent chemoselectivity and regioselectivity in me-tabolism of substrate. If NbaA would reduce otherwise the 4-nitrogroup of 2,4-DNBA to produce 4-hydroxylamino-2-nitrobenzoicacid, the 4-hydroxylamine group could be derivatized to the 4-(N-acetoxyacetamido) form, showing the molecular ion at m/z 282.2,because the described derivatization procedure completely acety-lates the 4-hydroxylamine group at both N and O centers (12).However, we detected no m/z 282.2 ion signal from the base peakchromatogram of 2,4-DNBA reduction. When derivatized withacetic anhydride, 2-hydroxylamino-4-nitrobenzoic acid appearedto preferentially undergo O-acetylation rather than N-acetylation,which formed N-acetylanthranilic acid (product I) from 2-hy-droxylaminobenzoic acid by the same procedure. Although N-acetylation is generally favored, some hydroxylamine groups ofnitroaromatic compounds are known to undergo O-acetylation(16). These chemical analyses show that NbaA specifically trans-forms 2-NBA and 2,4-DNBA to the 2-hydroxylamine compoundswith the consumption of NADPH.

FIG 5 (A) Size-exclusion chromatography and native PAGE analyses of NbaA oligomers during incubation of NbaA at pH 7.4 and 25°C. Molecular weight (Mr;in thousands) markers for size-exclusion chromatography were thyroglobulin (670,000), bovine-�-globulin (158,000), chicken ovalbumin (44,000), equinemyoglobin (17,000), and vitamin B12 (1,300). Native PAGE gels were analyzed with the column-loaded samples (denoted as load) and elution fractions (2 mleach) between 40 to 68 min. Molecular weight (Mr) markers for native PAGE were 175,000, 83,000, 62,000, and 48,000. Protein elution peaks and bands aredenoted with arrowed lowercase letters: a, homodimer; b, homotetramer; c, homohexamer; and d, homo-octamer. (B) Composition of NbaA oligomers (stackedbars) and their NADPH oxidation rates (line-symbol curves) with 1 mM 2-NBA (circles) or 2,4-DNBA (triangles) measured in 50 mM sodium phosphate buffer(pH 7.4) containing 1 mM NADPH, 0.1 mM MnCl2, and 10 �M FMN. (C) Measurements of NADPH oxidation rate by concentration of 2-NBA (open symbolson solid-line curves) or 2,4-DNBA (closed symbols on broken-line curves) after incubation for 0 (circles), 2 (triangles), 6 (squares), and 12 h (diamonds).

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Protein oligomerization. During incubation of NbaA at roomtemperature, 2-NBA- and 2,4-DNBA-reducing activities gradu-ally decreased as NbaA homodimer oligomerized to form higheroligomers, as observed by size-exclusion chromatography and na-tive PAGE analyses (Fig. 5A). In size-exclusion chromatography,both activities were overlapped in the elution fractions of theNbaA homodimer but not of higher oligomers. Decreasing levelsof NbaA homodimer during incubation were positively correlatedwith decreased NADPH turnover rates of 2-NBA and 2,4-DNBA(Fig. 5B). When enzyme kinetics for 2-NBA and 2,4-DNBA wereevaluated by the Hill equation, oligomerization of NbaA resultedin a decrease in the apparent maximum rates without significantlychanging the half-saturation constants and Hill slopes for sub-strate binding (Fig. 5C), suggesting that the protein oligomeriza-tion led to a decrease in enzyme concentration.

Intermolecular disulfide bond formation of NbaA. NbaAformed higher-order disulfide-bonded proteins under ambientconditions. The disulfide bond formation was similarly observedat 1 and 10 �M NbaA monomer concentrations, indicating a zero-order reaction independent of protein concentration (Fig. 6A).The disulfide bond formation occurred more rapidly at 37 than25°C. In addition, the intermolecular disulfide bond formation ofNbaA was largely increased by alkaline pH and H2O2 treatments

(Fig. 6B). Using various buffers with a pH range of 5.5 to 9.7, thepKa value of NbaA participating in the formation of intermolecu-lar disulfide bonds was estimated at approximately pH 7.4(Fig. 6C). At this point, the formation of intermolecular disulfidebonds causing enzyme inactivation was drastically increased byapproximately 7-fold from 3.4% h�1 below pH 7.4 to 24% h�1

above pH 7.4, as the pH was changed by one unit at a time. Thesedata indicate that NbaA can form inactive higher proteins throughthe formation of intermolecular disulfide bonds under physiolog-ical conditions.

SDS-PAGE and mass spectrometric analyses of disulfide-bonded isomers. SDS-PAGE under nonreducing conditionsshowed that NbaA formed three isoforms of each disulfide-bonded species (Fig. 7A). To analyze the disulfide bond positionsby mass spectrometry, the three isoform bands of dimer-sizedNbaA, considered the initial products of intermolecular disulfidebond formation, were excised from the gel. The trypsin-digestedisoforms resulted in different patterns of extracted ion chromato-grams for the 3 disulfide-bonded peptides derived from randomcross-linking of two tryptic peptides, 132ITDAPIAWEC*K142 and188LGGPNYC*R195 (Fig. 7B). The identities of these peptides wereconfirmed by tandem mass spectrometry (Fig. 7C). None of theother disulfide-bonded peptides theoretically derived from ran-dom cross-linking by the other cysteines at positions 39 and 103were detected. SDS-PAGE and mass spectrometry revealed thatcysteines at positions 141 and 194 mediate the formation of 3isoforms by random disulfide bonding.

Site-directed mutagenesis. To characterize the functionalroles of cysteines in NbaA, each cysteine (C39, C103, C141, andC194) was replaced with alanine by site-directed mutagenesis.Wild-type NbaA and the site-specific mutants (C39A, C103A,C141A, C194A, and C141A C194A) yielded different protein ex-pression levels (Fig. 8A, upper). Thus, it was necessary to deter-mine their molar concentrations in whole-cell extracts using theknown concentration of purified NbaA by Western blotting withanti-NbaA antibody (Fig. 8A, lower). The crude NbaA activity for2-NBA reduction (kcat � 84 � 1 s�1) decreased by approximately1.6-fold compared to that of purified NbaA (138 � 6 s�1), whereasthe crude enzyme activity for 2,4-DNBA (kcat � 47 � 1 s�1)increased by approximately 9-fold compared to that of purifiedNbaA (5.3 � 0.1 s�1). There was an inverse relationship be-tween enzyme activity and lag time such that the lag time for2,4-DNBA was markedly decreased from 396 to 75 s, whereas thelag time for 2-NBA increased from 20 to 29 s (Fig. 8B). As a neg-ative control, whole-cell extracts obtained from IPTG-inducedcells containing empty plasmid resulted in no detectible NADPHoxidation. It appeared that whole-cell extract contained certainfactors which enhance the enzyme activity for 2,4-DNBA at theexpense of its 2-NBA activity. Therefore, we used the crude en-zymes to accurately estimate the kinetic parameters, since a largeamount of active enzyme was inactivated over the prolonged lagtime.

C39A and C103A resulted in similar enzyme activities for2-NBA and 2,4-DNBA (Fig. 8C and D); they exhibited strongpositive cooperativity for 2-NBA (h, 2.8) and decreased the en-zyme activity for 2,4-DNBA (Table 3). These mutations were sim-ilar to the truncation of the V1 region, which markedly reducedthe enzyme activity for 2,4-DNBA but not 2-NBA (Fig. 2D), whileC141A and C194 showed different patterns for 2,4-DNBA and2-NBA. C141A decreased the enzyme activity for 2-NBA but not

FIG 6 Nonreducing SDS-PAGE of disulfide-bonded NbaA proteins. (A) Ef-fects of protein concentration (1 or 10 �M NbaA monomer) and temperature(25 and 37°C) at pH 7.0 for 1 h. A control sample (Ctrl) treated with 10 mMDTT at 60°C showed the reduction of disulfide-bonded proteins to NbaAmonomer. (B) Distribution patterns of NbaA monomer and higher disulfide-bonded proteins after 1 h of incubation with or without 10 mM H2O2 at pH 7or 10 at 25°C. (C) Relative levels of NbaA monomer (A1) and higher disulfide-bonded proteins (A2, A3, and A3) after 1 h of incubation at various pHconditions at 25°C. The pKa value was determined from a 10% decrease fromthe maximum level of NbaA monomer by fitting to a logistic curve.

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for 2,4-DNBA. This mutation did not significantly change theenzyme affinity to 2-NBA but enhanced the cooperativity for 2,4-DNBA. It appeared to enhance the specificity toward 2,4-DNBA atthe expense of the 2-NBA activity. In addition, C194A could re-duce the 2-NBA activity to a certain degree without a significantchange in the 2,4-DNBA activity. However, the double C141AC194A mutation diminished both 2-NBA and 2,4-DNBA activi-ties. These effects of C141A and C194A on the enzyme activitieswere similar to the differential specificity of NbaA which occurredduring disulfide bond formation under aerobic conditions. Thisindicated that oxidative modifications on either C141 or C194 canalter the catalysis and specificity of the enzyme, and that furthermodifications of both residues could lead to inactivation of theenzyme, as did the disulfide bond formation. Based on the presentdata, we propose that NbaA exists as a transient intermediate be-tween the reduced and oxidized forms of the four cysteines, andthis is responsible for modulating the catalysis and specificity.

DISCUSSION

We purified and characterized 2-nitrobenzoate 2-nitroreductase(NbaA) from P. fluorescens strain KU-7; NbaA specifically trans-forms 2-NBA and 2,4-DNBA into the 2-hydroxylamine com-pounds. Phylogenetic analysis suggests that NbaA belongs to a

new family of the flavin-containing reductase-like superfamily.Homologous genes are narrowly distributed in soil- and plant-associated agrobacteria, rhizobia, bradyrhizobia, and pseu-domonads and often comprise two or more homologous genes inthe genomic DNA and plasmids of some bacteria. The coexistenceof multiple homologous genes is likely associated with horizontalgene transfer and might be related to niche-specific adaptations todifferent types of plants and soils (17). The specific properties ofthese gene products have not been characterized. In this study, wefound that NbaA is able to reduce 2,4-DNBA as well as 2-NBA.2,4-DNBA is formed by photolysis of the explosive 2,4,6-trinitro-toluene (18). Some bacteria were able to degrade 2,4-DNBA in anitroaromatic pesticide-contaminated environment (19). How-ever, no specific enzyme for 2,4-DNBA reduction has been iden-tified. To our knowledge, this is the first report that NbaA is able tospecifically reduce 2,4-DNBA as well as 2-NBA.

NbaA binds with a divalent cation and FMN cofactor. TheFMN cofactor is required for catalysis; divalent cations, includingMn2�, Ca2�, and Mg2�, are not necessary for catalysis, rather theyincrease the reaction rate by reducing the lag time of the enzymaticactivity. The divalent cation-induced effects on NbaA are similarto those of glutamine synthetases in E. coli and Mycobacterium

FIG 7 (A) Patterns of disulfide-bonded NbaA protein isomers in nonreducing SDS-PAGE denaturing conditions. Mr indicates Bio-Rad Precision Plus proteinstandards in thousands (Lot 161-0375). (B) Extracted ion chromatograms (XIC) of precursor ions of 3 different disulfide-bonded dipeptides at a charge state (z)of �2 or �3. (C) Tandem mass spectra of 3 disulfide-bonded dipeptides. The assigned b and y ions generated by collision-induced fragmentation are labeled onthe one-letter amino acid sequences above the spectra, and the major fragment ions are shown in the tandem mass spectra.

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smegmatis (20, 21). When the kcat/K0.5S values are measured forMn2�-bound enzyme, the value for 2-NBA (110 �M�1 min�1) ismuch higher than that for 2,4-DNBA (0.44 �M�1 min�1). This isbecause the kcat value for 2,4-DNBA decreases and K0.5S increases

after a prolonged lag time of 690 s, while the 2-NBA activity has alag time of only 24 s. The kinetic behavior of NbaA for the 2substrates could not be described by the substrate-induced fitmodel (22) and relevant conformational changes in E. coli argini-

FIG 8 (A) Expression patterns of wild-type NbaA and the cysteine-to-alanine mutants. The lower panel shows molar concentrations of the crude enzymes inwhole-cell extracts determined using the known concentrations of purified NbaA by Western blotting (WB). (B) Continuous spectrophotometric assays for thecrude NbaA (0.1 �M monomer concentration) with addition of 1 mM 2-NBA or 2,4-DNBA to 50 mM sodium phosphate buffer (pH 7.4) containing 1 mMNADPH, 10 �M FMN, and 0.1 mM MnCl2 under aerobic conditions. The same protein amount extracted from empty plasmid-transformed cells was used toshow the absence of NADPH oxidation with 2-NBA or 2,4-DNBA under the same conditions. (C and D) 2-NBA (C) and 2,4-DNBA (D) kinetic curves ofwild-type NbaA and the cysteine-to-alanine mutants. Each 0.1 �M monomer of the crude enzymes was used in the reactions.

TABLE 3 Kinetic parameters of wild-type NbaA and cysteine-to-alanine mutant NbaA for 2-NBA and 2,4-DNBA

NbaA type

Parametera result for:

2-NBA 2,4-DNBA

vmax (�M min�1) K0.5S (�M) h vmax (�M min�1) K0.5S (�M) h

Wild-type 252 � 46 602 � 133 1.75 � 0.22 141 � 1 644 � 3 6.75 � 0.19C39A 252 � 9 231 � 12 2.87 � 0.42 7.3 � 3.4 624 � 225 3.34 � 2.34C103A 252 � 15 137 � 16 2.81 � 0.82 23.9 � 5.4 493 � 203 1.25 � 0.27C141A 149 � 29 417 � 94 2.19 � 0.57 145 � 2 435 � 17 15.3 � 6.9C194A 184 � 11 149 � 22 5.85 � 2.56 160 � 8 568 � 18 5.89 � 1.00C141A C194A 61 � 1 265 � 4 6.54 � 0.5147 50.1 � 4.6 565 � 38 4.26 � 0.91a Kinetic parameters estimated by the Hill equation fitted to the kinetic data shown in Fig. 8C and D are expressed as the estimated means � SD. vmax, apparent maximum rate;K0.5S, half-saturation constant for substrate binding; h, Hill coefficient.

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nosuccinate synthetase (23), taurine/alpha-ketoglutarate dioxy-genase (24), membrane insertase YidC (25), and multidrug trans-porter EmrE (26). The large difference in lag times forequilibration of enzymatic activity toward 2-NBA and 2,4-DNBAand failure to reduce 2,4-DNBA in the presence of DTT or underanaerobic conditions suggest a redox-induced conformationalchange of the enzyme specific to 2,4-DNBA.

Cysteines are vulnerable to oxidation and can cross-linkthrough disulfide bonds to other cysteines in close proximity, evenunder mild conditions (27). It is a rarely used amino acid thataccounts for about 2% in eukaryotic proteins and about 1% inprokaryotic proteins (28). Locations of some cysteines in the cat-alytic sites are highly conserved (referred to as motifs) in iron-sulfur cluster-containing proteins (29), thioredoxin-like redoxproteins (30), heme-binding proteins (31), and peroxiredoxinand flavoprotein reductase (32). Oxidation of such cysteines car-ries out biological functions in electron transfer, covalent sulfurbridge, and thiol-disulfide exchange reactions and triggers redoxprocesses that control cell regulatory pathways and induction of avariety of proteins, including transcription factors, molecularchaperones, and protein tyrosine phosphatases (33). In addition,several molecular studies of noncatalytic cysteines have shed lighton the functional role in protein stability, protein folding andrefolding, regulation of reactive oxygen signal transduction, andsubstrate-binding affinity and activity of enzyme (34–38). Thecommon mechanism underlying cysteine oxidation is exerted viaconformational changes through the formation of disulfide or cy-clic sulfenamide covalent bonds or sulfenic or sulfonic acids. Forexample, Bacillus subtilis ResA, a thioredoxin-like protein, is ableto undergo a redox-induced conformational change between thereduced and oxidized states and uses alternative conformations toselect the substrates (39). We indicate that alanine substitution ofthe four cysteine residues in NbaA at positions 39, 103, 141, and194 alters the catalytic properties and substrate specificity of theenzyme for 2-NBA and 2,4-DNBA. This demonstrates that oxida-tive modifications on the cysteine residues of NbaA can induce thedifferential specificity for the two substrates, 2-NBA and 2,4-DNBA. The oxidation of either C39 or C103, located around theV1 region, may reduce the enzyme activity for 2,4-DNBA but not2-NBA. On the contrary, the oxidation of either C141 or C194 inthe V2 region may reduce the enzyme activity for 2-NBA but not2,4-DNBA and may further modifications to form intermoleculardisulfide bonds that inactivate the enzyme.

A prolonged lag time for 2,4-DNBA provides no catalytic ad-vantage, since a large amount of the enzyme is inactivated throughthe formation of disulfide bonds. It is similar to treatment with athiol-blocking agent (e.g., DTNB) or a thiolate-complexing agent(e.g., ZnCl2). Size-exclusion chromatography and gel electropho-resis show that the NbaA homodimer is the basic catalyst, and thatthe enzyme is inactivated by the formation of higher oligomers viathe formation of intermolecular disulfide bonds. Mass spectrom-etry proves the formation of three isoforms of disulfide-bondedproteins by random disulfide cross-linking between the two cys-teines at positions 141 and 194. The random disulfide cross-link-ing of proteins with two cysteines is likely to increase the structuralcomplexity of the oligomer population by the large asymmetry ofisoform distribution. The rate of in vitro intermolecular disulfidebond formation of NbaA is markedly enhanced at ambient con-ditions above pH 7.4 or under oxidizing conditions. These factorsare critical for regulating cytosolic protein by oxidative modifica-

tions under physiological conditions (40, 41). In cells, some redoxproteins, such as the peroxiredoxin system and thioredoxin sys-tem, catalyze reversible oxidation and reduction reactions be-tween cysteines and disulfide bonds in cytosolic protein (41–43).Actually, the intermolecular disulfide bond formation of NbaAwas markedly increased by the exposure of E. coli cells to oxidativestress, while it was significantly reduced by a forced expression ofthioredoxin A (11). Therefore, the catalytic activity and substratespecificity of oxygen-sensitive NbaA may be more effectively con-trolled with such redox systems in cells.

In conclusion, we presented here that NbaA is a specific en-zyme for transformation of 2-NBA and 2,4-DNBA to the 2-hy-droxylamine compounds. However, it does not react with 2-NBAand 2,4-DNBA simultaneously. The mutation studies on the twovariable regions and cysteine residues support that the NbaA ac-tivities for the two substrates are modulated by oxidative modifi-cations of the four noncatalytic cysteines, which may induce someconformational changes in the substrate-binding sites. The con-formational change induced by reversible oxidative modificationof noncatalytic cysteine is superficially effective to regulate sub-strate availability by allosteric regulation of both catalysis andspecificity (37, 44). Further studies are needed to explore the redoxproperties of the cysteinyl residues and the mechanisms underly-ing the oxidative posttranslational modifications which lead toconformational changes in catalysis and specificity of the enzyme.

ACKNOWLEDGMENTS

This research was supported by the Marine and Extreme Genome Re-search Center Program of the Ministry of Land, Transport, and MaritimeAffairs, South Korea, and by the Next-Generation BioGreen 21 Program(SSAC grant PJ009025), Rural Development Administration, South Ko-rea. C.L. was supported by the Proteogenomic Research Program(2012M3A9B9036679) funded by the Korean Ministry of Education, Sci-ence and Technology, South Korea.

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