Functional and Structural Characterization of the Arylamine N-Acetyltransferase from the Opportunistic Pathogen Nocardia farcinica

doi:10.1016/j.jmb.2008.08.035 J. Mol. Biol. (2008) 383, 549–560

Available online at www.sciencedirect.com

Functional and Structural Characterization of theArylamine N-Acetyltransferase from the OpportunisticPathogen Nocardia farcinica

Marta Martins1†, Benjamin Pluvinage1†, Inès Li de la Sierra-Gallay2,Florent Barbault3, Julien Dairou1,4, Jean-Marie Dupret1,4

and Fernando Rodrigues-Lima1,4⁎
1Laboratoire de Cytophysiologieet Toxicologie Cellulaire(EA 1553), Université ParisDiderot-Paris 7, 75005 Paris,France2Centre National de la Recherche(CNRS FRC550), Institut deBiologie Physico-Chimique,75005 Paris, France3ITODYS, Université ParisDiderot-Paris 7, 75005 Paris,France4UFR des Sciences du Vivant,Université Paris Diderot-Paris7, 75013 Paris, France
Received 18 June 2008;received in revised form13 August 2008;accepted 14 August 2008Available online22 August 2008

*Corresponding author. EA 1553, UDiderot-Paris 7, 75005 Paris, Francfernando.rodrigues-lima@univ-pari† M.M. and B.P. contributed equaAbbreviations used: NAT, arylam

N-acetyltransferase; INH, isoniazid;sulfamethoxazole; AcCoA, acetyl coreading frame; DTNB, 5,5′-dithio-biHDZ, hydralazine; AMV, 4-aminoveData Bank; PBS, phosphate-buffered

0022-2836/$ - see front matter © 2008 E

ArylamineN-acetyltransferase (NAT) enzymes are found in a broad range ofeukaryotes and prokaryotes. There is increasing evidence that NATenzymescould contribute to antibiotic resistance in pathogenic bacteria such asMycobacterium tuberculosis. Nocardia farcinica is an opportunistic humanpathogen that causes pulmonary infections (nocardiosis) with clinicalmanifestations that resemble tuberculosis. While the genomic sequence ofthis prokaryote has been determined, studies of N. farcinica proteins remainalmost nonexistent. In particular,N. farcinica proteins putatively involved inantibiotic resistance mechanisms have not been described structurally orfunctionally. Here, we have characterized a newNATenzyme (NfNAT) fromN. farcinica at the structural and functional level. NfNAT is the first N.farcinica protein for which a 3D structure is reported. We showed that thisnovel prokaryotic isoform is structurally and functionally related to themycobacterial NAT enzymes. In particular, NfNAT was found to displayhigh N-acetyltransferase activity towards several known NAT substratesincluding the antitubercular drug isoniazid. Interestingly, isoniazid is notused for the treatment of nocardiosis and has been shown to be poorly activeagainst several nocardial species. On the contrary, NfNATwas found to bepoorly active towards sulfamethoxazole, a sulfonamide drug considered asthe treatment of choice for the treatment of nocardiosis. The functional andstructural data reported in this studywill help to develop our understandingof the role of NAT enzymes in nocardia and mycobacteria and may help inthe rational design of NAT antagonists for a range of clinical applications.

© 2008 Elsevier Ltd. All rights reserved.

Keywords: arylamine N-acetyltransferase; Nocardia farcinica; mycobacteria;antibiotics; structure
Edited by R. Huber
Introduction

Arylamine N-acetyltransferases (NATs; EC2.3.1.5) are xenobiotic-metabolizing enzymes that

niversité Parise. E-mail address:s-diderot.fr.lly to this work.ineSMX,enzyme A; ORF, opens-2-nitrobenzoic acid;ratrole; PDB, Proteinsaline.

lsevier Ltd. All rights reserve

catalyze the N-acetylation of arylamines, hydra-zines, and their N-hydroxylated metabolites bytransfer of the acetyl group of acetyl coenzyme A(AcCoA).1,2 The NAT enzyme kinetics proceeds viaa ping-pong bi–bi mechanism, in which a thioace-tyl enzyme intermediate is formed at the active-sitecysteine.3,4

NAT was first identified in humans as a key en-zyme involved in the inactivation of the antituber-culosis drug isoniazid (INH).5 Since then, NATenzymes have been found and characterized inseveral eukaryotic and prokaryotic species.2,6–8

Advances in the understanding of the structureand the functions of NATs have initially come fromthe determination of the X-ray structures of theenzyme from Salmonella typhimurium (StNAT).9

d.

550 Characterization of NAT from N. farcinica

Structures of prokaryotic NAT enzymes from Myco-bacterium smegmatis (MsNAT),10 Pseudomonas aerugi-nosa (PaNAT),11 Mesorhizobium loti (MlNAT1),12 andMycobacterium marinum (MmNAT)13 have now beenreported. These studies identified a common fold forNAT enzymes (NAT fold) that comprises threedomains (an N-terminal α-helical bundle, a centralβ-barrel, and a C-terminal α/β lid over the activesite) and a cysteine protease-like catalytic triad (Cys-His-Asp). Very recently, the NMR structure of Syrianhamster NAT214 and the crystal structures of the twohuman NAT enzymes (NAT1 and NAT2) have beendescribed.15 These studies showed that, contrary tobacterial NATs, an additional loop is present be-tween the second and third domains of Syrianhamster NAT2 and human NAT enzymes. Althougheukaryotic and prokaryotic NAT isoforms share thesame overall fold and a conserved active-site geo-metry, differences at the amino acid level are knownto have functional consequences by imparting diffe-rent aromatic amine substrate specificity and proteinstability to NAT enzymes.11,14–18Several bacterial NATs have been shown to

acetylate various aromatic amine substrates includ-ing antibiotics.2,19,20 Although the role of NAT inprokaryotes is unclear, it has been suggested thatthese enzymes may contribute to adaptative and/ordefense mechanisms towards environmental toxinspresent in the different habitats of bacteria.19,21 N-Acetylation of the sulfonamide antibiotic sulfa-methoxazole (SMX) by a NAT enzyme from Bacillusanthracis has been shown to contribute to the resis-tance of this bacterium to SMX.22 More importantly,the involvement of humanNAT2 in the polymorphicinactivation of INH has focused attention towardsMycobacterium tuberculosis NAT (MtNAT) and itsputative role in INH resistance. Several comprehen-sive studies based on MtNAT and on other myco-bacterial NAT enzymes clearly indicate that theseenzymes are involved in the resistance to the majorantitubercular antibiotic INH.2,23,24 Given the globalemergence of drug resistance in tuberculosis,MtNAT appears as a new putative target for deve-loping antitubercular drugs.25

Nocardia farcinica is a Gram-positive aerobic actino-mycetes that causes lung infections primarily inimmunocompromised individuals. Since the clinicalmanifestations of pulmonary infection due to N.farcinica (nocardiosis) resemble tuberculosis, nocar-diosis is often misdiagnosed as tuberculosis.26 Therecently completed genomic sequence of N. farcinicahas clearly demonstrated that this pathogen is close-ly related to M. tuberculosis and suggests that thesetwo bacteria are derived from a common ancestor.27

Biochemical and functional studies of N. farcinicaproteins remain essentially nonexistent;28 however,due to the many similarities of this bacterium to M.tuberculosis, such studies might be pertinent to ourunderstanding of both nocardiosis and tuber-culosis.27 Here, we describe the functional andstructural characterization of the NAT enzyme fromN. farcinica. NfNAT is the first N. farcinica protein tobe characterized at the structural level. Our data

should help us to understand the role of NAT en-zymes in the two related nocardia and mycobacter-ium genera and complement current work aimed atidentifying small-molecule inhibitors as bacterialNAT antagonists.2

Results and Discussion

Sequence analysis of the NAT open readingframe (ORF) of N. farcinica

Human infection by the opportunistic pathogenN.farcinica is increasingly diagnosed as a cause ofpulmonary disease in immunocompromisedpatients.26,29 The complete genomic sequence of N.farcinica IFM 10152 has been recently reported,27 andits analysis clearly showed that this pathogen wasclosely related to M. tuberculosis.27 These data sug-gested that the genomic and clinical similarities ofN.farcinica to M. tuberculosis could help understandtuberculosis.27 However, despite the clinical impor-tance of N. farcinica, few studies have been carriedout to characterize the proteins of this organism.28

Screening of the N. farcinica genome indicated thepresence of a unique ORF putatively encoding a 293-amino-acid NAT enzyme (NfNAT) (Genbank entry:YP_118052). Contrary to the NAT gene of M. tuber-culosis, which has been predicted as part of a six-geneoperon,30 we found that the NfNAT gene was notpredicted to be in a particular cluster (data notshown). NfNAT was aligned with the prokaryoticNAT enzymes that have been structurally (exceptMtNAT) and functionally characterized (StNAT,MtNAT, MsNAT, PaNAT, MlNAT1, and MmNAT)(Fig. 1).2,13 NfNAT was found to possess all of thewell-defined NAT functional motifs7,33 including theconserved NATcatalytic triad (Cys-His-Asp) (Fig. 1).Amino acid sequence alignment showed that NfNATshared sequence identities ranging from 28% to 35%with the other NAT enzymes. Despite differences inlength, NfNAT (293 amino acids) was found to bemore closely related to the mycobacterial isoformssuch as MtNAT (283 amino acids; 33% identity withNfNAT) or MsNAT (275 amino acids; 35% identitywith NfNAT) (Fig. 1). NfNAT and other prokaryoticNAT isoforms were subjected to phylogenetic analy-sis to examine the possible evolutionary relationshipsamong these enzymes (Fig. 2). NfNATwas includedin a monophyletic clade that contained MtNAT,MbNAT, MmNAT, MaNAT, and MsNAT, indicatingthat these sequences derived from a common ances-tor enzyme. The results obtained with NAT sequen-ces were in agreement with the taxonomic andgenomic studies done on N. farcinica, which showeda close phylogenetic relationship between this bacte-rium and M. tuberculosis.27 These data strongly sug-gest that NfNAT may have functions in commonwith MtNAT and support the concept that a com-prehensive characterization ofNfNATmight providean increased understanding of both nocardiosis andtuberculosis in humans.

Fig. 1. Multiple sequence alignment of NfNAT with selected prokaryotic NAT enzymes. Alignment of NfNAT with the sequence from well-characterized prokaryotic NATenzymes was made using ClustalW31 and ESPript32 programs. Red and yellow backgrounds indicate identical and highly similar amino acid residues, respectively. Stars indicateamino acids from the catalytic triad Cys82, His119, and Asp136 (numbering from NfNAT sequence). Conserved NAT functional motifs are indicated by the gray bars below thesequence alignment. The secondary structure of NfNAT was deduced from its crystal structure (PDB entry: 3D9W) and is represented above the sequence alignment. M.tuberculosis, MtNAT; M. marinum, MmNAT; S. typhimurium, StNAT; M. smegmatis, MsNAT; P. aeruginosa, PaNAT; and M. loti, MlNAT1.

551Characterization

ofNATfrom

N.farcinica

Fig. 2. Phylogenic analysis of NfNAT. The tree was obtained by the neighbor-joining method with multiple alignmentof protein sequence (ClustalW) using the TOP tool (DNA Data Bank of Japan) as described in Materials and Methods.Distance matrices were calculated using the Dayhoff substitution model. Ba, B. anthracis, Cc, Caulobacter crescentus; Ec, E.coli; Ma, Mycobacterium avium; Mb, Mycobacterium bovis; Ml, M. loti; Mm, M. marinum; Ms, M. smegmatis; Mt, M.tuberculosis; Na, Novosphingobium aromaticivorans; Nf, N. farcinica; Pa, P. aeruginosa; St, S. typhimurium.


Purification of the recombinant NfNAT enzyme

NfNAT protein was produced in Escherichia coliand purified as an N-terminal hexahistidine tag. Asshown in Fig. 3a, a highly purified NfNAT proteinwas obtained with a molecular mass consistent withthe predicted value (36 kDa). Western blot experi-ments (Fig. 3b) with a specific antibody againstMtNAT34 showed cross-reactivity with NfNAT. Asdiscussed above (Figs. 1 and 2), these results providefurther support for the suggestion that NfNAT is

closely related to mycobacterial NAT enzymes andin particular to MtNAT.

Characterization of the substrate profileof NfNAT

To test whether NfNATwas enzymatically active,we analyzed its catalytic activity and substrate pro-file. Specific activities of NfNAT towards aromaticamine substrates were determined with the 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) assay.35 To

Fig. 3. Expression and purifica-tion of recombinant NfNAT protein.(a) SDS-PAGE analysis of recom-binant NfNAT protein. PurifiedNfNAT (4 μg, Lane 1) was subjectedto SDS-PAGE under reducing condi-tions and stained with CoomassieBlue. (b) Purified humanNAT1 (1μg,lane 1) and NfNAT (0.4 μg, lane 2)were subjected to SDS-PAGE underreducing conditions, transferred to anitrocellulose membrane, and stain-ed with Ponceau S. (c) NfNAT wasimmunodetected using a polyclonalantibody raised against MtNATenzyme (1/1000 dilution).

Fig. 4. Comparison of the sub-strate specificity of NfNAT enzymewith other bacterial NATs. NfNATspecific activity towards severalprototypic NAT substrates was de-termined as described in Materialsand Methods and compared withliterature data (obtained under thesame conditions) for other bacterialNATs, namely, M. smegmatis(MsNAT), P. aeruginosa (PaNAT), S.typhimurium (StNAT), and M. mar-inum (MmNAT).11,13,35 Data forNfNAT and substrate abbreviationsare reported in Table 1. Errors fortriplicate values were a maximum of±10%.

Table 1. NfNAT activity towards typical aromatic NATsubstrates

Class/Compound Short name Rate (nmol min−1 mg−1)a

Arylamine drugs5-Aminosalicylate 5-AS 24,631Sulfamethazine SMZ 15Sulfamethoxazole SMX 29

Other arylamines4-Aminobenzoic acid pABA 1114-Aminosalicylate 4-AS 3722-Aminofluoreneb 2-AF 13,0274-Aminobiphenyl 4-AB 24,913Benzidine BZ 52,325Beta-naphthylamine BNA 23,734

Alkoxyanilines4-Aminoveratrole AMV 79,390

Hydrazine drugsIsoniazid INH 2878Hydralazine HDZ 87,761

NfNATactivity towards prototypic NATsubstrates was estimatedby measuring the rate of hydrolysis of AcCoA (nmol min−1 mg−1)in the presence of purified recombinant NfNAT, AcCoA (400 μM),and aromatic substrates (500 μM) as described previously.35

a Errors for triplicate values were a maximum of ±10%.b Assay was done in the presence of 5% (v/v) dimethyl

sulfoxide.

553Characterization of NAT from N. farcinica

allow direct comparison with substrate selectivityprofiles reported previously for other well-charac-terized prokaryotic NAT enzymes (StNAT, MsNAT,PaNAt, and MmNAT), we performed the assay forNfNAT under the same conditions.11,13 As shown inFig. 4 and Table 1, NfNAT was found to acetylateefficiently a broad range of aromatic substrates suchas hydralazine (HDZ), 4-aminoveratrole (AMV),and INH, which have been identified as goodMtNAT substrates.13,23 The rates of acetylation ofHDZ, AMV, and INH by NfNAT were found to bemuch higher than the rate reported for mycobacte-rial NAT enzymes (MsNAT and MmNAT). For ins-tance, HDZ was acetylated 3 and 12 times morerapidly by NfNAT than by MmNAT and MsNAT,respectively. For AMV, acetylation rates were 87 and26 times higher with NfNATwhen compared to thetwomycobacterial NATs. This overall trend was alsofound with the other aromatic substrates (except for2-aminofluorene, for which NfNAT and MmNATspecific activities were similar) and clearly indicatesthat NfNATexhibits in vitro specific activities greaterthan those of recombinant MmNAT and MsNAT. Inaddition, NfNAT was able to acetylate several sub-strates such as HDZ and AMV 2–3 times more effi-ciently than PaNAT, a prokaryotic isoform currentlyconsidered to be the most efficient NAT enzyme.11Overall, although the specific activity profile ofNfNAT differs slightly from that of MmNAT andMsNAT, NfNATappears to be a particularly efficientmycobacterium-related NAT enzyme. Differences inspecific activities between the closely related myco-bacterial MmNAT and MsNAT have also been re-ported13 and suggest that the role of NAT is likely todiffer depending upon the organism in which it isfound.36

Analysis of the acetylation of INH and SMXby NfNAT

Although the functions of prokaryotic NATs arelargely unknown, recent studies have shown thatcertain bacterial NAT enzymes could be involved inresistance mechanisms to antibiotics.2,22 In particu-lar, mycobacterial NATs such asMtNATandMsNAT

have been clearly shown to acetylate INH to itsinactive metabolite and to concomitantly decreasethe sensitivity of these mycobacteria to INH.2,23,34

The activity of NfNAT towards INH was furtherinvestigated by determining the Michaelis–Mentenparameters Vm,app and Km,app and the catalytic effi-ciency (Vm,app/Km,app ratio). NfNAT was found tohave aVm,app of 10,800±770 nmol min−1 mg−1 and aKm,app of 1414±167 μM, thus giving a catalytic effi-ciency of ∼7.6 nmol min−1 mg−1 μM−1 towardsINH. When compared to the parameters previouslyreported for MsNAT (Vm,app=115±33 nmol min−1mg−1; Km,app=87±18 μM; Vm,app/Km,app=1.3 nmolmin−1 mg−1 μM−1), it appears that, in vitro, NfNAThas a catalytic efficiency towards INH ∼6 timeshigher than that of MsNAT.10 These data stronglysuggest that NfNAT could contribute to INH insen-sitivity in N. farcinica as shown for the MsNAT orMtNAT enzymes.2 Generally, INH is not used to

Table 2. Data collection and refinement statistics

CollectionWavelength (Å) 0.931Space group P21Unit-cell parameters

a, b, c (Å) 61.34, 140.92, 74.85β (°) 101.830

Resolution (Å) 50–2.7 (3–2.7)Total number of reflections 177,129Total number of unique reflections 33,425Multiplicity 5.3 (5.34)Rmerge

a 15.5 (52.7)Mean I/σ(I) 12.63 (3.74)Overall completeness (%) 97.7 (94.7)

RefinementResolution (Å) 45.7–2.7Reflections (working/test) 31,753/1672Rcryst/Rfree

b 19/25.1Number of protein atoms 2265RMSD

Bond lengths (Å) 0.007Bond angles (°) 1.4

Ramachandran analysis (%)Most favored 82.9Allowed 100

Values in parentheses are for the highest-resolution shell.a Rmerge =∑h∑i|Ihi− ⟨Ih⟩|/∑h∑iIhi, where Ihi is the ith

observation of the reflection h, while ⟨Ih⟩ is the mean intensityof reflection h.

b R-factor=∑||Fo|−|Fc||/|Fo|. Rfree was calculated with asmall fraction (5%) of randomly selected reflections.


treat nocardiosis, particularly N. farcinica infec-tions,29 and this antibiotic has been reported to bepoorly active in a number of nocardial species.37

Although sensitivity of bacteria to INH is likely to begoverned by multiple mechanisms,38 the expressionof a NATenzyme that efficiently acetylates INH inN.farcinica may contribute to its relatively low sensi-tivity to this antibiotic. Further studies are needed toaddress this point. The treatment of choice for N.farcinica infections and, more broadly, for nocardio-sis is based on sulfamide antibiotics, in particularSMX.26,29,39 SMX and several other sulfamide anti-biotics are known to be biotransformed throughNAT-dependent acetylation.40 It has been suggestedrecently that a B. anthracis NAT isoform (BaNATC)could contribute to the resistance of this bacterium toSMX.22 In vitro, NfNATwas found to acetylate SMXwith a catalytic efficiency (Vm,app/Km,app=70 pmolmin−1 mg−1 μM−1) 10 times lower than that reportedfor the BaNATC isoform.22 Whereas the highcatalytic efficiency of NfNAT towards INH supportsits potential involvement in INH sensitivity of N.

Fig. 5. X-ray crystal structure of NfNAT. (a) Fold and surfarepresentation of the catalytic residues cysteine (C82), histidisequence). α-Helices are represented in red, β-strands in yellStructural alignment of NfNATwith other well-characterizedNfNAT is shown in red, StNAT in blue, MsNAT in green, andMomitted. Catalytic residues (C82-H119-D136) of NfNATare showmost important backbone structure differences between proka2), RII (the β-turn between β-strands 6 and 7), RIII (the β-turn bstrands 11 and 12).

farcinica, our data indicate that NfNAT is less likely toalter SMX therapeutic effects in N. farcinica. Theseresults support the notion that the role of NAT inprokaryotes may differ from one species to anotherdepending on substrate profile.36

Crystal structure of NfNAT

Crystallization studies were carried out to furthercharacterize NfNAT at the molecular level. Determi-nation of the structure of NfNAT may help us tounderstand the role of NAT in N. farcinica and morebroadly in other prokaryotes, in particular myco-bacteria. Such 3D structures may be of importance toidentify a specific subsite to target in drug design.13

The structure of NfNATwas solved at a resolution of2.7 Å (Table 2 and Fig. 5a). Despite low sequenceidentity, the X-ray crystallographic structure ofNfNAT revealed that this enzyme shares an almostidentical fold with that of other prokaryotic NATs,2,9

in particular MsNAT and MmNAT.10,13 The overallNfNAT structure consists of three domains of equi-valent size. The first two domains, a helical bundle(amino acids 1–98) and a β-barrel (amino acids 99–199), are disposed in such a way that three residues(Cys82, His119, and Asp136) form a catalytic triad.The third domain is linked to the second through aninterdomain helix (α6: amino acids 200–210). Asshown for other prokaryotic NAT structures,2 theinterface formed between domains 2 and 3 forms asubstantial active-site cleft. NfNAT has an RMSD of1.5 Å over 224 residues with MmNATand 1.7 Å over237 residues with MsNAT (Fig. 5b). Structural iden-tity with the other prokaryotic NAT structuresranged from 1.6 Å over 205 residues with MlNAT1to 2.6 Å over 171 residues with StNAT (Fig. 5b).These data highlight the close structural similaritiesbetween prokaryotic NATenzymes and in particularbetween NfNAT, MsNAT, and MmNAT. Compari-sons between prokaryotic NAT structures have pre-viously revealed small conformational differencesresulting from both single point insertions and dele-tions, which are accommodated within loops andform mobile loop regions linking more stable secon-dary structures.12 The largest and potentially mostsignificant deviations between NfNAT and otherprokaryotic NAT enzymes center on four regions(Fig. 5b). Indeed, regions RI (loop between strandsβ1 and β2, amino acids Gly105-Ala120), RII (β-turnbetween strands β6 and β7, amino acids Thr169-Asp175), RIII (β-turn between strands β7 and β8,amino acids Gly183-Gly186), and RIV (β-strandsβ11 and β12, amino acids Thr239-Ala253) were

ce. Structure is shown as ribbon trace with ball-and-stickne (H119), and aspartate (D136) (numbering from NfNATow, and coils in green. Surface is shown in light gray. (b)bacterial NATs. Structures are represented in ribbon trace.mNAT in yellow. For clarity, the structure of MlNAT1 wasn in ball-and-stick representation. Regions that display theryotic NATs are labeled RI (loop between β-strands 1 andetween β-strands 7 and 8), and RIV (the β-turn between β-

Fig. 5 (legend on previous page)


Fig. 6. INH docking. (a) Docking of INH at the catalytic site of NfNAT. Arginine 115 is shown in ball-and-stickrepresentation. (b) Docking of INH at the catalytic site of MsNAT. Proline 105 is represented in ball-and-stickrepresentation (numbering from MsNAT sequence).


found to be the regions that undergo significantbackbone movement when prokaryotic NAT struc-tures were compared. Moreover, temperature fac-tors/mobility (B-factors) analysis indicated thatthese regions display mainly high B-factor values(N45 Å) when compared to the average mean B-factor of NfNAT (28 Å). Interestingly, inM. marinum,these regions were found to be flexible and to un-dergo significant movements upon binding toCoA.13 In addition, several amino acid positions inthese four regions have been shown to be involvedin either AcCoA/CoA or INH binding.13,41 Overall,these data support the proposition that structuraldifferences in these four regions among prokaryoticNATenzymes may contribute to differences in enzy-matic properties of NAT enzymes. Albeit small,such structural differences between NAT enzymescould be promising to design small inhibitorycompounds.13,15

Analysis of INH binding by in silico docking

In silico docking experiments were carried out tobetter understand the binding of INH to NfNAT. Tothis end, the NfNAT structure reported in this studyand the published co-crystal structure of MsNATwith INH [Protein Data Bank (PDB) entry: 1W6F]were used for the initial steps of the docking proce-dure and for comparison. These experiments (Fig. 6)indicated that the mode of binding of INH to NfNATwas similar to the one reported forMsNAT.41 Indeed,INH was found to bind to the active site of NfNATmainly through interactions mediated by aminoacids conserved in the two enzymes (Phe51, Cys82,

Val107, Thr118, Phe139, and Phe218 in NfNAT).However, the free energy of binding (ΔGbind) ofINH to MsNAT was found to be lower than thatdetermined for NfNAT (−17.6 kcal/mol for MsNATand −2 kcal/mol for NfNAT). Accordingly, theaffinity (Km,app) of MsNAT for INH was reported tobe around 100 μM,10 whereas NfNAT binds INHwith a Km,app of 1414 μM. Structural analysissuggests that these differences in INH-bindingenergies (and subsequently in Km,app) betweenNfNAT (Fig. 6a) and MsNAT (Fig. 6b) are likelydue to the presence of an Arg residue in NfNAT(Arg115) instead of a Pro residue inMsNAT (Pro105).Weak electrostatic attraction between Arg115 andINH may explain higher ΔGbind and Km,app values.However, as discussed above, although the affinityof INH for NfNAT is lower than that for MsNAT,NfNAT has, in vitro, a catalytic efficiency for INH 6times higher than that ofMsNAT. This higher activityof NfNAT towards INH relies on a higher catalyticturnover (higher Vm,app) compared to that ofMsNAT.10 This may be due to a better stabilizationof transition states during catalysis.42

Nocardiosis due to N. farcinica is on the rise.26

Although the genomic sequence of this pathogen hasbeen determined,27 N. farcinica proteins (and inparticular those putatively involved in antibioticresistance mechanisms) have not been describedstructurally or functionally. Here, we have characte-rized functionally and structurally a new NAT en-zyme (NfNAT) fromN. farcinica. NfNAT is the firstN.farcinica protein for which a 3D structure is nowavailable. These data are expected to improve ourunderstanding of the role of NAT enzymes in nocar-


dia and mycobacteria and may be exploited todesign NAT antagonists for a range of clinicalapplications.

Materials and Methods

Materials

4-Aminosalicylic acid, 5-aminosalicylic acid, sulfa-methazine, SMX, 2-aminofluorene, HDZ, INH, para-ami-nobenzoic acid, AcCoA, DTT, DTNB (or Ellman's reagent),and His-Select Nickel-NTA superflow resin were obtainedfrom Sigma-Aldrich. The bacterial expression vectorpET28a was purchased from Novagen. The Bradfordprotein assay kit was supplied by Bio-Rad. All otherreagents were purchased from Sigma-Aldrich. Polyclonalantibody against NAT from M. tuberculosis was a kind giftfrom Prof. Edith Sim (OxfordUniversity, UK) and has beendescribed elsewhere.34

Sequence alignment and phylogenetic analysis

Protein sequences were retrieved from the NationalCenter for Biotechnology Information GenPept database.Amino acid alignments were performed using ClustalW1.8.31 The neighbor-joining phylogeny was generated withthe Dayhoff substitution matrix using TOP (DNA DataBank of Japan‡).

Molecular cloning and plasmid construction

DNA fromN. farcinica IFM 1015227 was kindly providedby Dr. J. Ishikawa (National Institute of Infectious Disease,Tokyo, Japan). The nfnat ORFwas amplified by PCR usingthe following primers: 5′ CCCGGATCCATGAGCAAG-CCCGACGAC 3′ (sense) and 5′ AAGCGGCCGCCTA-CGCGGTGCCCGCAGC 3′ (reverse), with BamHI andNotI restriction sites shown, respectively, in boldface.High-fidelity DNApolymerase (Pfu) was used for the PCRcorresponding to a first denaturation step (94 °C, 3 min),followed by 40 cycles of annealing (55 °C, 1.5 min),extension (72 °C, 1 min), denaturation (94 °C, 1 min), and,finally, an extension step (72 °C, 5 min). The product wascloned into a pET28a expression vector for expression andpurification as a 6His-tagged recombinant protein. DNAsequencing was used to check the insert sequences.

Protein expression and purification

The pET28a-nfnat plasmid was transformed into E. coliBL21(DE3)pLys cells for production and purification of theprotein. Briefly, transformed bacterial cells were grown at37 °C for 5 h in the presence of 0.2 mM IPTG. Cells (1 l)were harvested by centrifugation (5000g, 4 °C, 10 min) andwashed with phosphate-buffered saline (PBS). Aftercentrifugation, the pellet was resuspended in 15 ml ofcold PBS containing lysozyme (1 mg/ml final concentra-tion) and protease inhibitors. After 30 min of incubation at4 °C, 0.1% Triton X-100 was added for a further 1 h ofincubation at 4 °C. The lysate was subjected to sonicationon ice (4 pulses of 10 s each) and pelleted (12,000g, 30 min).

‡http://www.ddbj.nig.ac.jp/search/about-e.html

The supernatant was incubated with 1.5 ml of His-SelectNickel resin for 2 h at 4 °C in the presence of 20 mMimidazole. Resin was poured into a column and washedwith PBS (containing 20 mM imidazole and 150 mMNaCl). Recombinant NfNAT protein was eluted with300 mM imidazole in PBS (plus 150 mM NaCl). Theeluates were reduced with 10 mM DTT for 10 min at 4 °Cbefore dialysis against 25 mM Tris–HCl, pH7.5, and 1 mMethylenediaminetetraacetic acid. The purity of the enzymewas assessed by SDS-PAGE followed by Coomassie Bluestaining. The recombinant protein was stored at −80 °C.

Protein determination, SDS-PAGE, and Westernblot analysis

Protein concentrations were determined using theBradford assay (Bio-Rad). For electrophoresis, sampleswere combined with reducing 4×SDS sample buffer,separated on 10% SDS-PAGE gels and stained with Amidoblack. For Western blot analysis, separated proteins wereelectrotransferred onto a nitrocellulose membrane. Themembrane was blocked by incubation with Tris-bufferedsaline/Tween 20 (TBS) supplemented with 5% nonfat milkpowder (diluted in TBS) for 1 h. The membrane wasincubated for 2 h in TBS (1% nonfat milk) with antibodiesraised against M. tuberculosis NAT (1:1000).33 Followingincubation with a secondary conjugated antibody (anti-rabbit at 1:100,000) and washing of the membrane, ECLreagent (Amersham Pharmacia) was used for detection.

Enzyme assays and determination of kineticsparameters

The DTNB (or Ellman's reagent) assay was used tomeasure the AcCoA-dependent acetylation of prototypicaromatic NAT substrates by the NfNAT enzyme asdescribed by Brooke et al.35 Briefly, purified recombinantproteins and arylamine substrates (500 μM final) weremixed in a 96-well ELISA plate and preincubated (37 °C,5 min) in assay buffer (25 mM Tris–HCl, pH7.5, and 1 mMethylenediaminetetraacetic acid). To start the reaction, weadded AcCoA (400 μM final). Total reaction volume was100 μl. The reaction was quenched with 25 μl of DTNB(5 mM) in guanidine hydrochloride buffer (6.4 M guani-dine–HCl and 0.1 M Tris–HCl, pH7.3). A plate reader wasused tomeasure the absorbance at 405 nm. All assays weredone in triplicate under conditions giving a linear initialrate. Controls were carried out in the absence of enzyme orAcCoA. The amount of CoA in the reaction was deter-mined by comparison with a standard curve obtainedusing DTNB. Data were expressed as the means±SD.Apparent kinetic parameters (Vm,app and Km,app) for

SMX and INH (0–2 mM) in the presence of 400 μMAcCoAwere calculated from nonlinear regression analysis asdescribed previously.22 For ping-pong bi–bi mechanisms(as for NAT enzymes), apparent Vm/apparent Km ratiosare equal to true kinetic parameter ratios Vm/Km.

43

Apparent Vm/apparent Km ratios were therefore used tocompare catalytic efficiencies towards the different aro-matic substrates tested as reported previously.21

Crystallization and data collection

Crystallization trials of NfNATwere performed at 294 Kwith EasyXtal Tool X-Seal (Qiagen) using the hanging-drop vapor-diffusion method. Using a mix of 2 μl ofprotein (10 mg/ml) with 1 μl of reservoir solution, we


obtained crystals in two conditions: 15% polyethyleneglycol 8000, 0.8 M sodium chloride, and 0.1 M N,N-bis(2-hydroxyethyl)glycine, pH8.5 (condition 1); 4.7% polyethy-lene glycol 8000, 0.133 M sodium chloride, and 0.1 M 4-morpholineethanesulfonic acid, pH6 (condition 2). Wecharacterized and collected data up to 2.4 Å from the twoconditions (Table 2).X-ray data were collected from cryo-cooled crystals

using 20% glycerol as cryoprotectant. The diffraction datawere collected at beamline ID14-3 of the EuropeanSynchrotron Radiation Facility. Crystals grown from thefirst crystallization condition belonged to space group P21and those grown from the second condition belonged tospace group P212121, with four molecules per asymmetricunit in both cases. Diffraction data were processed usingthe program XDS.44 Relevant data collection statistics arereported in Table 2.The structure of NfNAT protein was solved at 2.7 Å

resolution by molecular replacement, using the coordi-nates of M. loti NAT1 enzyme (PDB entry: 2BSZ). Therefinement data are given in Table 2.

Docking studies

INHwas constructed with the SYBYL 7.2 software45 andgeometrically optimized using Tripos force field. The INH/NfNAT complex structure was obtained by fitting theNfNAT coordinates to the INH/MsNAT complex8 (PDBcode: 1W6F) and by adding the extracted ligand coordi-nates to the NfNAT structure. The protein/ligand struc-tures were geometrically optimized with the Amber 9software.46 Ligand parameters were created according tothe antechamber procedure and the partial charge evalua-tion following the AM1 bond charge correction me-thod.47,48 The general Amber force field49 was set for theligand whereas the ff03 force field50 was used for theprotein. Minimizations were donewith 2000 steps of steep-est descent followed by 3000 steps of conjugate gradient.Solvent effects were taken into account through a general-ized Born/implicit solvent model.51 The MolecularMechanics/Poisson–Boltzmann Surface Area method52

allowed for the evaluation of the ligand/protein interactionfree energies as detailed elsewhere.53

Accession code

Atomic coordinates and structure factors informationhave been deposited in the PDB with accession code3D9W.

Acknowledgements

This work was funded by grants from the Minis-tère de l'Enseignement Supérieur et de la Recherche,Leg Poix (Chancelerie des Universités de Paris),Association pour la Recherche sur le Cancer, Asso-ciation Française contre les Myopathies, and Caissed'Assurance Maladie des Professions Libérales-Provinces. M.M. is supported by a PhD fellowshipfrom the Ministère de l'Enseignement Supérieur etde la Recherche. B.P. is supported by a PhD fel-lowship from the Délégation Générale pour l'Arme-ment. We would like to thank Professor Edith Sim

(Oxford University, UK) for the anti-MtNAT anti-body and for helpful discussions. Wewould also liketo thank the Ministère des Affaires Etrangères etEuropéennes (Partenariat Alliance). We acknowledgethe European Synchrotron Radiation Facility forprovision of synchrotron radiation facilities and wewould like to thank Ganesh Natrajan for assistancein using beamline ID14-3.

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Documents

Functional and Structural Characterization of the Arylamine N-Acetyltransferase from the Opportunistic Pathogen Nocardia farcinica