Identification of the Enzyme Responsible for N-Acetylation ofNorfloxacin by Microbacterium sp. Strain 4N2-2

Dae-Wi Kim,a,b Jinhui Feng,a* Huizhong Chen,a Ohgew Kweon,a Yuan Gao,c Li-Rong Yu,c Vanessa J. Burrowes,a* John B. Sutherlanda

Divisions of Microbiologya and Systems Biology,c National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, USA; Department ofBiotechnology (BK21 Program) and Institute of Microbiomics, Chung-Ang University, Anseong, Republic of Koreab

Microbacterium sp. 4N2-2, isolated from a wastewater treatment plant, converts the antibacterial fluoroquinolone norfloxacin toN-acetylnorfloxacin and three other metabolites. Because N-acetylation results in loss of antibacterial activity, identificationof the enzyme responsible is important for understanding fluoroquinolone resistance. The enzyme was identified as glu-tamine synthetase (GS); N-acetylnorfloxacin was produced only under conditions associated with GS expression. The GSgene (glnA) was cloned, and the protein (53 kDa) was heterologously expressed and isolated. Optimal conditions and bio-chemical properties (Km and Vmax) of purified GS were characterized; the purified enzyme was inhibited by Mn2�, Mg2�,ATP, and ADP. The contribution of GS to norfloxacin resistance was shown by using a norfloxacin-sensitive Escherichiacoli strain carrying glnA derived from Microbacterium sp. 4N2-2. The GS of Microbacterium sp. 4N2-2 was shown to act asan N-acetyltransferase for norfloxacin, which produced low-level norfloxacin resistance. Structural and docking analysisidentified potential binding sites for norfloxacin at the ADP binding site and for acetyl coenzyme A (acetyl-CoA) at a cleftin GS. The results suggest that environmental bacteria whose enzymes modify fluoroquinolones may be able to survive inthe presence of low fluoroquinolone concentrations.

Fluoroquinolones are widely used as human and veterinary an-timicrobial agents (1). Because their persistence in the envi-

ronment (2) may act as a selective pressure for naïve strains toacquire resistance (3), an understanding of the fate of these drugsshould help to prevent drug resistance. The principal resistancemechanisms for fluoroquinolones include the following: (i) mu-tation of genes for the drug targets (GyrA, GyrB, and ParC) (4–6),(ii) mimicking the drug targets (QnrA, QnrB, and QnrS) (7–9),(iii) reduction of drug accumulation in the cells (AcrAB-TolC andQepA) (4, 10, 11), and (iv) enzymatic modification of the drug[N-acetylation by AAC(6=)-Ib-cr] (12–14).

In some strains of Escherichia coli, a mutated aminoglycosideacetyltransferase, AAC(6=)-Ib-cr, acetylates fluoroquinolones atthe N-terminal of the piperazine ring (12–14) and enhances bac-terial resistance to these drugs (14). Production of N-acetylnor-floxacin and N-nitrosonorfloxacin by environmental Mycobacte-rium sp. strains has also been found, but the enzymes responsiblefor modifications are unknown (15) and the aac(6=)-Ib-cr variantgene has not been detected in these strains (12).

A norfloxacin-modifying bacterium, Microbacterium sp. strain4N2-2, was isolated from wastewater (16). Some strains of thisgenus have been isolated from human clinical specimens, and themajority of them have shown fluoroquinolone resistance (17).Microbacterium sp. 4N2-2 transforms norfloxacin into four differ-ent metabolites, including N-acetylnorfloxacin, and cell extractsof this strain catalyze the N-acetylation of norfloxacin (16). N-Acetylation is enhanced by Casamino Acids and inhibited by am-monium and nitrate (16), suggesting that the enzyme responsiblefor norfloxacin N-acetylation might also be involved in nitrogenmetabolism.

In the present study, an enzyme with N-acetyltransferase activ-ity was isolated from Microbacterium sp. 4N2-2 and identified.The coding gene was cloned and heterologously expressed in E.coli for biochemical characterization of the enzyme and its contri-bution to norfloxacin resistance. Binding models of the enzyme

with norfloxacin and a cofactor were proposed by in silico struc-ture modeling and ligand binding (docking) analysis. The resultsprovide insight into the potential for enzymes of environmentalbacteria to contribute to drug resistance.

MATERIALS AND METHODSStrains, chemicals, and media. Microbacterium sp. strain 4N2-2 was iso-lated from a wastewater treatment plant in Little Rock, AR (16). E. coliBL21(DE3) pLysS, for heterologous overexpression of the target protein,was purchased from Novagen (EMD Millipore, Billerica, MA). Bothstrains were stored in 20% glycerol at �80°C.

Norfloxacin (Sigma-Aldrich, St. Louis, MO) was prepared as a 10 mgml�1 stock solution in 40 mM KOH for cultures and enzyme assays.

OM medium (16) was used as the basal medium for cultures of Mi-crobacterium sp. 4N2-2. For extraction of proteins, cultures were grown inOM medium with 2.0 g liter�1 Casamino Acids instead of ammoniumand nitrate (16). To test the effects of nitrogen sources on N-acetylation,1.0 g liter�1 of glutamate, glutamine, or ammonium was substituted. Cul-tures of Microbacterium sp. 4N2-2 were incubated at 30°C with shaking at200 rpm for 9 days for protein extraction or for 10 to 17 days for N-acety-lation analysis.

LB broth with 10 g liter�1 NaCl (BD Biosciences, Franklin Lakes, NJ)was used for cultures of E. coli, and LB agar (20 g liter�1 agar) was used forthe norfloxacin disk assay. Cultures of E. coli were incubated aerobically at37°C with shaking at 200 rpm for 18 h.

Received 26 July 2012 Accepted 23 October 2012

Published ahead of print 26 October 2012

Address correspondence to John B. Sutherland, john.sutherland@fda.hhs.gov.

* Present address: Jinhui Feng, Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences, Tianjin, China; Vanessa J. Burrowes, Department ofEnvironmental Health, Emory University, Atlanta, Georgia, USA.

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

doi:10.1128/AEM.02347-12

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High-performance liquid chromatography. After cultures were cen-trifuged (13,000 � g) and filtered (0.45 �m pore size), 10 �l of the spentmedium was directly injected for high-performance liquid chromatogra-phy (HPLC) analysis of norfloxacin N-acetylation at 280 nm as previouslydescribed (16).

N-Acetylation by cell extracts. After incubation in OM medium withCasamino Acids for 10 to 17 days, cells of Microbacterium sp. 4N2-2 werewashed twice and suspended in 50 mM HEPES buffer (pH 7.5) containing10% glycerol. A 4-ml volume of the cell suspension was disrupted by fivepassages through a French pressure cell at 137,000 kPa. Cell debris wasremoved by centrifugation (13,000 � g) and filtration (0.22 �m poresize). Various pH values, temperatures, and amounts of enzyme, sub-strate, and cofactors were used to evaluate the optimum conditions for thereaction. Cell extracts were incubated with 0.06 mM norfloxacin– 0.2 mMacetyl coenzyme A (acetyl-CoA) (14)–5.0 mM CaCl2–50 mM Tris-HClbuffer (pH 8.0) at 45°C. Production of N-acetylnorfloxacin was moni-tored by direct HPLC analysis of incubated reaction mixtures and con-trols.

Purification of the enzyme responsible for N-acetylation. Ammo-nium sulfate was added to cell extracts to reach a final concentration of 1.0M; the mixture was centrifuged and filtered (0.45 �m pore size). Thefiltrate was applied to a HiPrep phenyl FF 16/10 column (GE Healthcare,Piscataway, NJ). The proteins were eluted using 60 ml of 1 M (NH4)2SO4,60 ml of 0.5 M (NH4)2SO4, 60 ml of 0.2 M (NH4)2SO4, and 60 ml of water.The active fractions [eluted by the use of 0.2 M (NH4)2SO4], as shown byHPLC, were collected, concentrated, and diluted in 25 mM piperazine-HCl buffer (pH 5.0). The fractions were applied to a Q XL column (GEHealthcare). The proteins were then eluted using 60 ml of 25 mM phos-phate buffer, 60 ml of a linear gradient to 0.2 M NaCl, 60 ml of 0.5 M NaCl,and 60 ml of 1 M NaCl. The fractions with N-acetylation activity eluted by0.5 M NaCl were collected and applied to a Mono Q HR 5/5 column (GEHealthcare). The proteins were eluted with a linear gradient of 0 to 1 MNaCl.

Protein identification by mass spectrometry. Proteins from theMono Q column were separated on an SDS-PAGE gel (18). The bandswere excised, destained with 25 mM NH4HCO3–50% acetonitrile, cutinto smaller pieces, and dried in vacuo. Proteins were then digested withtrypsin (Promega, Madison, WI)–25 mM NH4HCO3 (pH 8.3) at 37°C for16 h. The resulting peptides were extracted with aqueous 70% acetoni-trile–5% formic acid by the use of sonication and lyophilized.

The tryptic peptides from each gel band were analyzed by reversed-phase nanoflow liquid chromatography–tandem mass spectrometry(LC-MS/MS) (19). Briefly, the peptides were redissolved in 0.1% formicacid and injected onto a fused silica capillary electrospray ionization (ESI)C18 column (9 cm by 75 �m [inside diameter]), which was coupled onlineto an Orbitrap mass spectrometer (LTQ-Orbitrap XL; Thermo Electron,San Jose, CA). Mobile phases A (0.1% formic acid–water) and B (0.1%formic acid–acetonitrile) were delivered by a Dionex UltiMate 3000 Nanoand Cap LC system (Dionex Softron GmbH, Germering, Germany). Pep-tides were separated using a step gradient of 2% to 42% solvent B for 40min and 42% to 98% solvent B for 10 min at a flow rate of 250 nl/min. Themass spectrometer was operated in a data-dependent mode, in which theseven most abundant peptide molecular ions from an MS survey scan(acquired in the Orbitrap analyzer) were dynamically selected for colli-sion-induced dissociation (CID) and analyzed in the linear ion trap usinga normalized collision energy of 35%.

The raw data from tandem mass spectrometry were searched againstthe Microbacterium testaceum protein sequence database (20) of the Na-tional Center for Biotechnology Information (http://www.ncbi.nih.gov/).The SEQUEST cluster, running BioWorks (revision 3.3.1 SP1; ThermoElectron), was used for the identification of peptides and proteins.

Gene cloning. Genomic DNA of Microbacterium sp. 4N2-2 was ex-tracted with an UltraClean Microbial DNA isolation kit (MoBio Labora-tories, Carlsbad, CA) following the manufacturer’s instructions. Degen-erate primers (5=-GGM CAG CTB TTC GAY GGM TCV TCS ATC CG-3=

and 5=-GSA GCT CGT AGA GGT CCT TGT CGA YSG GYK C-3=) wereused for PCR. After the sequence of the amplicon had been obtained, twonew primers were prepared to amplify upstream and downstream se-quences to give the full sequence of the target gene. A random nonamerprimer (GE Healthcare) and a reverse primer for upstream (5=-GTC ACGTCG GGG ATG AGC TGC ATG TCG-3=) were used to amplify the startregion. A random nonamer primer and a forward primer for downstream(5=-TCG AAC CCG AAG GCC AAG CGC ATC GAG-3=) were used toamplify the stop region. Sequence fragments were assembled in silico togive the full nucleotide sequence.

Heterologous expression and purification of the protein. The for-ward primer (5=-CCG TTC CTC CAG GAG TTG ACA TAT GTT CACCAC C-3=) and reverse primer (5=-NNN NNN NGG ATC CTC ASA CSCCGW AGT ACA GCT-3=) for the pET-11a plasmid (Novagen) were de-signed to amplify the full target gene (glnA) from the genomic DNA ofMicrobacterium sp. 4N2-2 (underlined letters are recognition sequencesfor NdeI and BamHI, respectively). The sequence of the degenerateprimer region was confirmed by comparing it with the full gene sequenceobtained. The amplified target gene (glnA; 1,425 bp) was inserted at theNdeI and BamHI sites of pET-11a, and the expression construct was con-firmed by restriction enzyme analysis and sequencing. The expressionconstruct was then introduced into the E. coli BL21(DE3) pLysS expres-sion host to yield E. coli BL21(DE3) pLysS/pET-11a-glnA. Expression ofthe target protein was controlled by the T7lac promoter of pET-11a bygradual addition of isopropyl-�-D-thiogalactopyranoside (IPTG) (finalconcentration, 0.8 mM) to cultures in LB medium for 1 h at 28°C.

After induction, the cultures were incubated for 4 h, harvested bycentrifugation, washed twice with 10 mM phosphate buffer (pH 7.2), andcentrifuged again. After a freeze-thaw step, they were resuspended in thesame buffer. Cells from 1.5 liter were suspended in 25 ml of phosphatebuffer and disrupted by sonication in an ethanol-ice bath. Cell debris wasremoved by centrifugation and filtration. Ammonium sulfate was gradu-ally added, to a final concentration of 20% to 100%, to find the optimumconcentration. The precipitate not containing the target protein, as shownby a norfloxacin N-acetylation assay and SDS-PAGE analysis, was pelletedand discarded.

The target protein was then precipitated from the supernatant withmore ammonium sulfate. The pellet was resuspended in 10 mM Tris-HCl(pH 7.8) and dialyzed with the same buffer to 20 ml, using a Centriconcentrifugal filter unit (EMD Millipore). A HiPrep Q XL 16/10 column (GEHealthcare) was used for purification with a gradient of 0 to 2.0 M NaCl in10 mM Tris-HCl (pH 7.8) at a flow rate of 1 ml min�1. The active fractionswere concentrated and dialyzed with 20 mM Tris-HCl (100 mM NaCl, pH7.5) to 5 ml. The fractions were applied in sequence to a gel filtrationcolumn (HiLoad 26/600 Superdex 200 prep grade; GE Healthcare). Thetarget protein was eluted with 20 mM Tris-HCl (100 mM NaCl, pH 7.5) ata flow rate of 2 ml min�1. Thyroglobulin (669 kDa), ferritin (440 kDa),aldolase (158 kDa), and conalbumin (75 kDa) from GE Healthcare wereused as standards.

The eluted protein was concentrated and dialyzed in 50 mM HEPESbuffer (pH 7.5) with 10% glycerol. Purification was monitored by SDS-PAGE analysis, and protein was analyzed by using a MicroBCA proteinassay kit (Thermo Scientific).

Norfloxacin resistance as shown in broth cultures and a disk assay.Overnight cultures of E. coli BL21(DE3) pLysS/pET-11a-glnA in LB me-dium were transferred into 200 �l of fresh LB medium as a 1% inoculum.Cells were grown at 28°C in 96-well plates with agitation in a microplatereader (Synergy 2; BioTek, Winooski, VT) at 28°C. When the opticaldensity at 600 nm had reached 0.2, IPTG was added at a final concentra-tion of 0.8 mM and norfloxacin was added at 0, 0.5, 1.0, 2.0, 5.0, 10.0, and20.0 �g ml�1. Growth was monitored every 15 min up to 16 h. All testswere repeated in triplicate.

For the disk assay, cells cultured overnight were spread with a sterilizedcotton swab on LB agar, with or without 0.8 mM IPTG. A norfloxacin disk(Remel, Lenexa, KS) (10 �g) was applied to the middle of each plate. After

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15 h at 28°C, the diameter of the clear zone was measured. All tests wererepeated in triplicate.

Enzyme activity analysis and inhibitor screening. Enzyme activitywas analyzed with 0.06 mM substrate– 0.2 mM acetyl-CoA–5 mMCaCl2–50 �l of 50 mM Tris-HCl buffer–20 mM NaCl (pH 8.0). Oneenzyme unit was defined as the amount of enzyme that converts 1 �mol ofnorfloxacin to N-acetylnorfloxacin, with saturated acetyl-CoA, at pH 8.0and 45°C in 1 h.

To screen potential inhibitors of N-acetylation activity, glutamate,glutamine, NH4Cl, MgCl2, MnCl2, ATP, and ADP were prepared as 100mM stock solutions and added to the reaction mixture at final 2 mMconcentrations. Activity of the enzyme was expressed as percent residualactivity compared to that of control reaction mixtures without inhibitors.

In silico docking analysis. A three-dimensional (3-D) protein struc-ture of the gene, based on the structure of the glutamine synthetase (GS)from Mycobacterium tuberculosis (21), was generated using the SWISS-MODEL server (22). PDBsum (http://www.ebi.ac.uk/pdbsum/) provideda quick overview, including schematic diagrams and interaction of GSwith ligands (ADP, norfloxacin, and acetyl-CoA). Automated docking ofnorfloxacin and acetyl-CoA to GS was performed using AutoDock Vina(23). The grid box (Center_x � �25.368, Center_y � 83.074, Center_z �11.188) was centered in the catalytic active region (size_x � 58, size_y �50, and size_z � 56). The maximum number of binding modes to gener-ate was 1,000 for all docking analyses. An input pdbqt file of GS, withnorfloxacin having the lowest binding energy of �8.0 kcal mol�1, wasused for the AutoDock docking acetyl-CoA simulation. Initial attempts tochoose the functional binding modes of norfloxacin and acetyl-CoA wereguided by (i) the nucleotide ADP binding site and (ii) the distance (�5 Å)between the N-terminal of the piperazine ring and the S1P of acetyl-CoA.PyMOL (0.99RC6) (http://www.pymol.org/) was used to visualize allstructural figures.

RESULTSPartial purification and identification of protein responsible fornorfloxacin N-acetylation. The N-acetylation activity in Micro-bacterium sp. 4N2-2 increased for up to �7 to 9 days in OMmedium with Casamino Acids (Fig. 1), so cell extracts were pre-pared from cultures grown in this medium for 9 days. Acetyl-CoA(14) was found to be required as an acetyl group donor for N-acetylation by cell extracts, and CaCl2 was found to be required asa cofactor.

To identify the responsible protein, selected bands containingpartially purified protein (�50 to 55 kDa) were excised from an

SDS-PAGE gel and analyzed by MS/MS. Eight proteins were con-sidered to be possibly responsible for N-acetylation of norfloxa-cin, but glutamine synthetase (GS) was considered the most likelybecause N-acetylation activity in Microbacterium sp. 4N2-2 is gov-erned by the nitrogen source and is strongly enhanced by Casa-mino Acids (16). Expression of GS is also enhanced by CasaminoAcids in Bacillus subtilis (24). Casamino Acids lacks glutamine(the product of GS, which causes feedback inhibition), but gluta-mate (the substrate) represents 8.4% to 15.9% of the total aminonitrogen (BD Bionutrients Technical Manual; BD Science).

To understand the relation between GS and norfloxacin N-acetylation, cultures of Microbacterium sp. 4N2-2 were grownwith various nitrogen sources. Production of N-acetylnorfloxacinwas found in media with Casamino Acids or glutamate but not inmedia containing glutamine or ammonium (Fig. 1).

Cloning of gene encoding N-acetyltransferase. The gene en-coding GS (glnA) was cloned for heterologous expression andthen characterized biochemically to reveal any unexpected func-tions of the protein. In the absence of a genome database for Mi-crobacterium sp. 4N2-2, degenerate PCR primers were used forcloning. Genes encoding GS (with the open reading frame [ORF]numbers from annotated genomes) from Microbacterium testa-ceum StLB037 (MTES3453), Clavibacter michiganensis subsp.sepedonicus (CMS1619), Leifsonia xyli CTCB07 (LXX10080), Ar-throbacter phenanthrenivorans sphe3 (Asphe15760), Renibacte-rium salmoninarum ATCC 33209 (RSal2447), and Mycobacteriumsmegmatis mc2155 (MSMEG4290) were aligned and used for de-signing degenerate primers. The forward primer (nucleotides 145to 173 of MTES3453) and the reverse primer (nucleotides 1,183 to1,212 of MTES3453) were used to generate an amplicon (1.1 kbp)that was used subsequently to obtain sequence information for themiddle region of glnA. Two other primers (a reverse primer forupstream and a forward primer for downstream), designed fromthe sequence information obtained, were used to amplify regionsoutward from the middle fragment of glnA. The fragments wereassembled manually to give a full nucleotide sequence for glnA ofMicrobacterium sp. 4N2-2.

The cloned gene sequence, which was deposited in GenBank(JX901058), shared 86%, 82%, 81%, 73%, 73%, and 65% deducedamino acid sequence identities, respectively, with the strains usedfor primer design, whereas GS from E. coli K-12 shared only 54%identity with the deduced protein. Two other GSs of M. testaceumStLB037 (MTES3446 and MTES1058) shared 36% and 30% iden-tities, respectively, with the deduced protein.

Comparison of MS/MS data with the sequence of GS. Afterthe cloning of glnA, peptide information for the GS of Microbac-terium sp. 4N2-2 was sought in the MS/MS spectra measured pre-viously during partial purification of the proteins. One suggestedpeptide sequence, based on the M. testaceum StLB037 genome,was IPITGSNPK. The corresponding deduced amino acid se-quence of GS of Microbacterium sp. 4N2-2 was IPLTGSNPK; theMS/MS spectrum of this peptide was exactly identified (P �0.001) in the data. This implied that GS from the cell extract ofMicrobacterium sp. 4N2-2 was most likely the protein responsiblefor N-acetylation.

Heterologous expression of GS and activity analysis. Basedon the sequence obtained, an expression construct (pET-11a-glnA) was prepared and expressed in E. coli BL21(DE3) pLysS.Cells from IPTG-induced and noninduced cultures of E. coli, con-taining either pET-11a-glnA or pET-11a, were harvested and dis-

FIG 1 Production of N-acetylnorfloxacin by Microbacterium sp. 4N2-2. Thenitrogen sources were Casamino Acids (�), glutamate (�), glutamine (Œ),and ammonium chloride (o). d, days.

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rupted. GS, which was expressed only in the IPTG-induced cellswith pET-11a-glnA, had the predicted size of �53 kDa (Fig. 2A).Cell extracts from these cultures showed high activity for convert-ing norfloxacin to N-acetylnorfloxacin, whereas the low activity(background) in cell extracts from the controls presumably orig-inated from E. coli enzymes.

Contribution of GS to norfloxacin resistance. Because Micro-bacterium sp. 4N2-2 grew well with a norfloxacin concentration ofmore than 70 �g ml�1, the contribution of GS to norfloxacinresistance was not evaluated directly in this strain. A norfloxacin-sensitive strain, E. coli BL21(DE3) pLysS, was used to show the roleof this enzyme in norfloxacin resistance. Under the conditionsused for this assay, the enzyme was active in the soluble fraction.

In broth cultures of E. coli BL21(DE3) pLysS pET-11a-glnAwithout IPTG, 0.5 �g ml�1 of norfloxacin inhibited growth (Fig.3). Induced cells expressing heterologous GS did not grow as wellas noninduced cells in the absence of norfloxacin, indicating thatthere was a metabolic load due to the heterologously expressedprotein. After induction of GS, the induced cells grew in the pres-ence of 0.5 and even 1.0 �g ml�1 norfloxacin at a rate similar to

that of induced cultures without norfloxacin. However, growth ofboth induced and noninduced cultures was inhibited by 2.0 �gml�1 norfloxacin (Fig. 3).

The norfloxacin disk diffusion assay also supported the ideathat GS contributed to norfloxacin resistance. The diameter of theinhibition zone for E. coli cells without glnA was not significantlyaltered by IPTG, but E. coli cells with glnA showed an �3-mm-smaller inhibition zone diameter (statistically significant; P �0.05) in IPTG-induced cultures than in noninduced cultures(Fig. 4).

Purification of heterologously expressed GS. GS was precip-itated with 55% ammonium sulfate and then sequentially purifiedwith an anion exchange column (Table 1). The molecular mass ofGS, calculated as 53.32 kDa from its deduced amino acid se-quence, coincided with the approximate size shown by SDS-PAGE (Fig. 2B). Gel filtration indicated a protein complex with anapproximate size of 640 kDa, implying that, like other GSs (25),this GS may form a homododecamer.

Characterization of GS. Using GS purified by ammonium sul-fate precipitation and anion-exchange chromatography, opti-

FIG 2 SDS-PAGE gels showing (A) heterologous expression of Microbacterium sp. 4N2-2 glutamine synthetase in the soluble fraction of E. coli BL21(DE3)pLysS/pET-11a-glnA with IPTG induction and (B) purification of glutamine synthetase of Microbacterium sp. 4N2-2 from heterologous overexpression.

FIG 3 Decreased sensitivity to norfloxacin (0 to 2 �g ml�1) due to induction of glutamine synthetase in E. coli BL21(DE3) pLysS/pET-11a-glnA. IPTG-induced(�) and noninduced (�) liquid cultures were used to monitor norfloxacin sensitivity by induction of glutamine synthetase in the E. coli BL21(DE3) pLysS host.

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mum conditions for norfloxacin N-acetylation were evaluated.The optimum pH and temperature were determined to be 8.0 and37 to 55°C, respectively. Acetyl-CoA at 0.2 mM was required as anacetyl group donor and calcium ion at 5.0 mM for enzyme activa-tion. With 0.2 mM acetyl-CoA, there was �5% abiotic back-ground in total production of N-acetylnorfloxacin at pH 8.0. Ap-parent Km and Vmax values were obtained from Lineweaver-Burkplots; with saturated acetyl-CoA, the Km was 2,208 �M and Vmax

was 13.2 mU (mg protein)�1.Glutamate, ammonia, and glutamine did not inhibit norfloxa-

cin N-acetylation by the purified enzyme, but ATP and ADPcaused 48% and 39% inhibition, respectively (Fig. 5). This indi-cated that norfloxacin does not bind to the pocket for substratesand products but instead binds to the ATP and ADP binding site(21, 26). Mg2� and Mn2�, which may activate GS for its usualsubstrates and coordinate the substrates at the active site (21, 27),instead caused 48% and 54% inhibition, respectively (Fig. 5).

In silico structural analysis. The 3-D structural model of GSfrom Microbacterium sp. strain 4N2-2 (Fig. 6A) was constructedusing the crystal structure (Protein Data Bank code 2BVC) of theGS from Mycobacterium tuberculosis (21) as a template. The rootmean square (RMS) deviation (C) of the GS model (474 aminoacids) superimposed on the 2BVC structure (486 amino acids)was 0.79 Å, and 471 amino acid residues were aligned with 63.4%

sequence identity. Figure 6A shows a biologically relevant dodec-amer model with two hexameric rings.

In GS, the active site between subunits has a cleft �14 Å inlength, �9 Å in width, and �11 Å in height. In the GS-ADP

FIG 5 Inhibition of N-acetylation activity of purified glutamine synthetasefrom Microbacterium sp. 4N2-2 by substrates and cofactors (2 mM each).

FIG 4 Norfloxacin disk assay of noninduced (gray bars) and IPTG-induced(white bars) cultures of E. coli BL21(DE3) pLysS containing plasmids with orwithout the glnA insert encoding glutamine synthetase. The assay was used tomonitor changes in norfloxacin sensitivity due to expression of glutaminesynthetase. In the cells with the glnA insert, the reduced diameter of the clearzones of inhibition due to induction of glutamine synthetase was statisticallysignificant (P � 0.05).

TABLE 1 Purification of recombinant glutamine synthetase from E.colia

Step

Totalprotein(mg)

Totalactivity(mU)

Specificactivity(mU/mg) Yield (%)

Crude cell extract 206.9 85.9 0.42 100.0Ammonium sulfate

precipitation35.0 73.4 2.10 85.4

Anion exchange 3.1 7.0 2.26 8.2a Activities were determined with acetyl-CoA and CaCl2.

FIG 6 Model of Microbacterium sp. 4N2-2 glutamine synthetase structure andpotential binding modes of norfloxacin and acetyl-CoA. (A) Top and side views ofglutamine synthetase complex. The dimeric active site is indicated by a whitesquare on the surface representation. (B and C) Electrostatic surface diagrams ofthe substrate binding pocket, colored by charge, and the potential binding modesof norfloxacin (B; five potential positions) and acetyl-CoA (C; 10 potentialpositions).

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complex, ADP is placed in almost the same manner in the cleft, asshown in the 2BVC structure (Fig. 6B). The AutoDock dockingnorfloxacin-GS simulation proposed 504 possible binding solu-tions. About 16 of them were located at the binding site of ADP, inwhich the reaction center (-N) of the piperazine ring lies appro-priately in the cleft. Figure 6B shows the five binding modes withthe lowest binding energy (��8.00 kcal mol�1), all of which havesimilar conformations and locations in a cleft of GS.

Figure 6C shows 10 potential functional binding modes ofacetyl-CoA with the lowest binding energies of ��6.00 kcalmol�1 from 687 possible binding solutions. As revealed in acloseup of the binding modes of acetyl-CoA (Fig. 6C), despite itsdispersed binding positions, most of the functional acetyl groupmoieties resided in the bottom of a cleft bound to the piperazinering of norfloxacin.

DISCUSSION

Many bacterial enzymes that N-acetylate various substrates useacetyl-CoA as an acetyl donor (28–30). In bacteria as well as hu-mans, N-acetylation may be involved in detoxification of drugs(28, 29, 31, 32), including fluoroquinolones (14). The activity ofGS in members of the Actinomycetales is regulated by transcrip-tional regulation and posttranslational adenylylation (33–35).The peptide sequence of GS was found in MS/MS spectra from thepartial isolation of proteins of Microbacterium sp. 4N2-2, and itexactly matched the deduced protein sequence for GS. When itwas heterologously expressed in E. coli and purified, GS alsoshowed norfloxacin N-acetylation activity.

Although N-acetylation of antibiotics by GS has not been pre-viously reported, there are reports of N-acetylation of proteins byGS. A so-called “moon lighting property” of GS in Mycobacteriumspp. is the N-acetylation of glutathione S-transferase, with a poly-phenolic acetate as an acetyl group donor (36, 37). The measuredactivity of Microbacterium sp. 4N2-2 GS for norfloxacin N-acety-lation was not as high as that for glutamine synthesis; neither wasit as high as the activities of other N-acetyltransferases (14, 36, 37),as shown by the Km and Vmax values. The Km for norfloxacin wassimilar to the Km for glutamate, but it was more than 10-foldhigher than the Km values reported for ATP and NH4Cl with theGS of Nocardia asteroides (38). High Km and low Vmax values maybe explained by an accidental substrate-enzyme relationship.Thus, the contribution of GS to resistance, which is effective onlyfor low norfloxacin concentrations, may be understood from thehigh Km and low Vmax.

Together with the biochemical observations, including (i) thelow enzyme activity, (ii) acetyl-CoA as an acetyl group donor, (iii)the relatively high Km for norfloxacin, (iv) the inhibition by ATPand ADP, and (v) the Ca2� requirement, the structural model alsoprovides insights into the N-acetylation activity of GS. This en-zyme not only requires acetyl-CoA and Ca2� for N-acetylation ofnorfloxacin, but it also has a structural environment suitable forfunctional binding of the substrates. When the predicted bindingwas examined with respect to the active site of the enzyme, thegeometry of the active site seemed unlikely to be problematic forbinding. Moreover, with norfloxacin bound in the active site, thepiperazine ring would orient toward the open space, which simul-taneously could accept the acetyl group of acetyl-CoA. As revealedin the docking analysis of the complex, when GS bound norfloxa-cin with the lowest binding energy (�8.0 kcal mol�1), most of theacetyl group of the bound acetyl-CoA was located in the bottom of

a cleft, placing the substrate in an ideal position for functionalgroup donation. The substrate binding modes and geometry ofthe active site support the idea that GS can join norfloxacin andacetyl-CoA in the active site. The N-terminal of the piperazinering lies near the reactive S1P atom of acetyl-CoA, which is anappropriate position for functional group transfer. Therefore, therelatively low N-acetylation activity of GS suggests that the reac-tion depends on the precisely coordinated functional binding ofthe substrates, norfloxacin and acetyl-CoA, and Ca2�.

The GS of Microbacterium sp. 4N2-2 appears to belong to afamily of highly conserved bacterial GSs (39). Other bacterial GSsthat acetylate fluoroquinolones may also exist, considering thatGS is conserved in actinobacteria with high structural homology(21, 39) and that N-acetylation activity for norfloxacin is found inseveral Mycobacterium spp. (15). This idea also is supported by theprotein N-acetylation function of GS of M. smegmatis (37).

The study of various GSs for N-acetylation activity, and en-zyme engineering based on models of protein-ligand binding,should provide insight into the N-acetylation function of GS.Moreover, because N-acetylation is an important detoxificationmechanism for bioactive molecules (40, 41), GSs of environmen-tal bacteria may be able to modify various chemicals, includingantibiotics.

In nutrient-limited and biofilm bacteria, the starvation re-sponse mediated by guanosine pentaphosphate is associated withtolerance to antibiotics, including fluoroquinolones (42). Expres-sion of GSs of E. coli, Bacillus subtilis, and Mycobacterium spp. isalso enhanced by the stringent response of nutrient starvation,especially with respect to ammonium (43–45). This implies that inoligotrophic sites or biofilms, a bacterial strain with a GS similar toMicrobacterium sp. 4N2-2 might show fluoroquinolone resis-tance. Most environmental sites have low-level (nanogram perliter) contamination by fluoroquinolones (46–48), so activationof GS by nutrient nitrogen limitation could contribute to fluoro-quinolone resistance.

This report describes the modification of a fluoroquinolone byan enzyme from an environmental bacterium. The approach usedin this research, including selective isolation of the bacterium,enzyme activity analysis, protein isolation and characterization,and structure modeling, represents a useful strategy for enzymeidentification and characterization in studies of biotransforma-tion and antibiotic resistance.

ACKNOWLEDGMENTS

We thank C. E. Cerniglia, M. Hart, and S. L. Foley for advice and helpfulcomments on the manuscript.

This research was supported in part by the appointments of D.-W.Kim and J. Feng to the Research Participation Program and that of V. J.Burrowes to the Summer Student Research Program at the National Cen-ter for Toxicological Research, administered by the Oak Ridge Institutefor Science and Education through an interagency agreement between theU.S. Department of Energy and the U.S. Food and Drug Administration.

The views presented in this article do not necessarily reflect those ofthe U.S. Food and Drug Administration.

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