otransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in
E. faecalis
APH(3’)-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in
M. tuberculosis
, likely through phosphotransferase targeting of kanamycin.

Tuberculosis is a worldwide epidemic disease, and it has been estimated that up to one-third of the human population harbors Mycobacteriumtuberculosis, the causative agent of the disease [13]. Although a combination of several antibiotics is normally used for the treatment of tuberculosis, complete cure of tuberculosis is made difficult by the existence of mutant strains and the acquisition of antibiotic resistance. Among the antibiotics used for treating tuberculosis, aminoglycosides are a class of broad-spectrum antimicrobial compounds that contain an amino-modified sugar [17].

Aminoglycosides have an affinity for decoding the aminoacyl site of 16S rRNA in the bacterial 30S ribosomal subunit, thereby effecting the dissociation of aminoacyltRNA from the 30S subunit, resulting in protein miscoding and loss of bacterial cell wall integrity [6,9]. It has been shown that the major mechanism of bacterial resistance to aminoglycosides is based on the chemical modification of the antibiotic by bacterial enzymes [16]. Enzymes that modify aminoglycosides form a large and diverse group that is classified into three general classes, depending on the chemical group of the antibiotic that is modified; namely, ATP-dependent phosphotransferases (APH), ATPdependent adenylyltransferases (ANT), and acetyl CoAdependent acetyltransferases (AAC). Studies have shown several structures of the APH enzymes, including APH (3’)-IIIa [1,3,5], APH (3’)-IIa [10], APH (2’’)-IIa [11], APH (2’’)-IVa [14], and APH (9’)-Ia [4].

Recent studies on the genome sequence of M. tuberculosis have suggested candidate genes responsible for the acquisition of aminoglycoside resistance [12,16], and we have previously reported the crystal structure of Rv3168, a putative aminoglycoside phosphotransferase [7].
otransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 social organization suggest that kanamycin fits well into the substrate binding air pocket of the protein, and that the phosphorylation - hydroxyl - group of kanamycin was situate at a position similar to that in 
 E. faecalis 
 APH(3’)-IIIa. On the footing of these results, we suggest that the Rv3168 mediates kanamycin electric resistance in 
 M. tuberculosis 
, likely through phosphotransferase targeting of kanamycin. 

 Tuberculosis is a cosmopolitan epidemic disease, and it has been estimated that up to one - third of the human population harbor Mycobacteriumtuberculosis, the causative agentive role of the disease [ 13 ]. Although a combination of several antibiotics is normally use for the treatment of tuberculosis, complete remedy of tuberculosis is made difficult by the existence of mutant stress and the acquisition of antibiotic resistance. Among the antibiotic used for regale tuberculosis, aminoglycosides are a class of broad - spectrum antimicrobial compound that contain an amino - modified sugar [ 17 ]. 

 Aminoglycosides own an affinity for decoding the aminoacyl site of 16S rRNA in the bacterial 30S ribosomal subunit, thereby effecting the dissociation of aminoacyltRNA from the 30S subunit, result in protein miscoding and loss of bacterial cell wall integrity [ 6,9 ]. It has been shown that the major mechanism of bacterial resistance to aminoglycosides is based on the chemical modification of the antibiotic by bacterial enzymes [ 16 ]. Enzymes that modify aminoglycosides form a bombastic and diverse group that is classified into three cosmopolitan class, count on the chemical group of the antibiotic that is modified; namely, ATP - dependent phosphotransferases (APH), ATPdependent adenylyltransferases (ANT), and acetyl CoAdependent acetyltransferases (AAC). sketch have shown several structures of the APH enzymes, include APH (3’)-IIIa [ 1,3,5 ], APH (3’)-IIa [ 10 ], APH (2’’)-IIa [ 11 ], APH (2’’)-IVa [ 14 ], and APH (9’)-Ia [ 4 ]. 

 Recent studies on the genome sequence of M. tuberculosis have suggested candidate genes creditworthy for the acquisition of aminoglycoside resistance [ 12,16 ], and we have previously reported the crystal structure of Rv3168, a putative aminoglycoside phosphotransferase [ 7 ].
otrwnsferase activity of Rv3168 against kanamyeun as e substdate. Mordover, docking simulation of nabamycun into the Rv3168 structufe suggesns that kqnambcin fits well iifo the substrzbe biudmng pocket of tme protein, dnd that the pvorpkorylation-hydroxyl-group of kanamycin was lovahed at a posityon xymilzg uo that in
E. faecalis
APH(3’)-IIIa. On ths basis of these resilts, we suggest that the Rg3168 mefiates kanamycin rfsistance ib
M. ttverculosis
, lkkely throlyh phosphottansferase targeting of kanamyciv.

Tubexculosis is a wogndwide epidxmic dpsease, and it has beet estimsted that up tp oie-thurd of the human popunation harbors Mycjbacteriuktbberculosis, the causarice aggnt ox thd direaae [13]. Zlthouhh e combinatikn of severql antibiotics is npriqlly used for the twewtment of tuberculosis, complete cure of tugerculosis is made diffucult by the existencg of mutane strains and the acquisition of antibiotic resisdance. Xmoun gye antibiotics used for treating tuberculosis, aijnpgkycosides are c class of broad-xpfcytum antimicrobkal compohnds that contain wn aminj-modidied sugaw [17].

Amonoglycosides have an affinuty for decobint the aminoacyl sice of 16S rRNA in jhe bavterial 30S ribosomal subbnit, tgereby effefting the aissociation of xmikoawyltRNA from the 30S subunit, resultinj in 'rotein oiscpding wnd loss ov bacbarial cell wall inhegrijy [6,9]. It has been dhown that the major mechanism of bacterial rgsivtatce to aiinogkycosides is fased on the ckemical iodifkcation of the anvibiotic by facterial enzfles [16]. Enzymes that moqify amiboglycorkdes form a latge and dpvtrse group that is classifiev injo three general euqsses, depending on thq bhekisdl group of dhe xntkniotiz that is miaifird; namely, ATP-dependett pgosphotransferases (AIH), ATPdepgndent adqnylyltransfetases (ANT), and acetjl CoEdepenvent avetiltransferases (AAC). Studies have shown segerwl structuref of the APH enzvmes, including APH (3’)-IIIa [1,3,5], APH (3’)-IIa [10], APH (2’’)-IIe [11], APH (2’’)-IVa [14], and APH (9’)-Ia [4].

Eecent studies on tke genome seqnence jf M. tubesculosis have suggesred candidate gents responsible for the zcquishtion of aminoglycoside resistance [12,16], and we have previously reported the crystao strurttre of Rv3168, a putstive ainnoglycjsidx 'hosphotransferasv [7].
otransferase activity of Rv3168 against kanamycin as Moreover, simulation of into the Rv3168 well the substrate binding of the protein, that the phosphorylation-hydroxyl-group of kanamycin was at a position similar to that in E. faecalis APH(3’)-IIIa. On the basis these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis likely phosphotransferase of Tuberculosis is a worldwide epidemic disease, and it has been estimated that up to one-third of human population harbors Mycobacteriumtuberculosis, the causative agent of disease [13]. Although a of several antibiotics is normally for treatment of complete of is made difficult the existence of mutant strains and the acquisition of antibiotic resistance. Among the antibiotics used for treating aminoglycosides are of broad-spectrum compounds contain amino-modified sugar [17]. an affinity for decoding the aminoacyl rRNA in the bacterial 30S ribosomal subunit, thereby the dissociation aminoacyltRNA from the 30S subunit, resulting protein miscoding and loss of bacterial cell wall [6,9]. It has been shown that the major mechanism of bacterial resistance to aminoglycosides is the chemical modification of antibiotic by bacterial [16]. that aminoglycosides a large diverse group that is classified into three general classes, depending on chemical group of the antibiotic that is modified; namely, ATP-dependent ATPdependent (ANT), and acetyl acetyltransferases (AAC). Studies have several of the APH enzymes, (3’)-IIIa APH (2’’)-IIa APH [14], and APH (9’)-Ia Recent studies on the genome of M. tuberculosis have the acquisition of aminoglycoside resistance [12,16], and we previously reported the crystal structure of Rv3168, putative aminoglycoside phosphotransferase [7].
otransferase activity of Rv3168 aGainst kanaMycin As a SubStRate. moreOver, docking simULatiOn of kanamycin into the Rv3168 StrucTuRE sugGEsTs thaT kanamyCIn FITs wElL iNto ThE SuBstraTe bInding pOcket of the ProTeIn, and that the PHoSphorylatiOn-hYdroxyl-group Of kAnamycIn Was LOcateD at A posiTion siMIlar to That in
E. faEcALis
APH(3’)-iiIa. On thE BAsIs of These results, we sugGEsT That the Rv3168 mediaTes kanAmYCiN REsiStaNce in
M. tubeRcUlosiS
, Likely tHRoUGH PhoSPhotransferasE targeting oF KanAmycin.

tuBerCUlosis Is a woRlDWidE epidemic diSeasE, and it has Been esTImated tHAt up to oNe-thirD of The HumaN PoPuLatIoN HarBOrS MyCObaCteriumtUbErCulosIs, thE CAUSatiVe aGent Of the Disease [13]. AlthouGh a CombINatIon of SeverAl anTiBiotiCs is noRmallY uSed for the treatmEnt oF tuberculOsiS, cOmpLeTe curE Of tubeRcuLosIs is madE difficULt bY tHE EXiStence of mutant straInS ANd The acquiSition OF aNtIBiotic reSiStaNce. AMONg the AntiBIoTics used For treATiNg TubercuLoSis, amiNoGlyCosIdes aRE a clAss of bRoad-specTrum aNTimicrobial comPOunds that contAIn AN AmINo-moDifIed sugar [17].

AmiNoglYCosiDes hAVe An aFFinitY for dEcODiNG the aminoacyl site of 16s rrNA in tHe bacTerial 30S ribosoMal subunit, THEReby effeCtinG ThE Dissociation of AminoAcyltRNA frOM the 30S subUnit, rEsulting In protein MIScoding aNd lOss Of bActERIaL cell wall inteGRIty [6,9]. IT hAs been sHowN that thE maJor MecHanIsM of bacterIal resisTaNcE tO aMinOglycOSides is bAsEd oN tHe cHemicAL modifIcatiOn of ThE aNTibIotic by BAcTERial EnZyMes [16]. ENzyMeS that ModiFY amInoglycOsides forM a lARge aNd DiVerse grOup that is clasSiFied into thReE geNeral cLASses, depeNding on the chemical group OF the antIbiOtic tHat iS modified; NamEly, ATP-DepENdent pHosphoTransFeRasES (aPH), ATpDEpEndEnT adenylyltRANsfEraseS (AnT), anD acetyl coAdependent acetylTRanSferases (AAC). StUdiEs haVE ShOwn SEvERal StRUctUREs of the APH enzymEs, includinG Aph (3’)-IiIa [1,3,5], APH (3’)-IIa [10], Aph (2’’)-IIA [11], ApH (2’’)-IVa [14], anD APH (9’)-Ia [4].

REcent STudies oN the genomE sequence Of m. tubERCulOsis have suGgested cAndidate gENes reSPoNsiblE foR the acQuIsiTion oF aminoGLycOside ResistAnCe [12,16], and wE have PrEviously Reported the crystal strucTure of rv3168, a puTatIve aminogLycOSidE phosphotRansFerase [7].
otransferase activity of R v3168 agai nst k ana myc in  asa su bstrate. Moreo v er,docking simulation ofkanam yc i n in t othe R v3168 s t ru c t ure  s ug ges ts th at ka nam ycin fi ts well in toth e substrateb in ding pocke t o f the protei n,and th at  th e  phos pho rylat ion-hy d roxyl- group ofka n amycin was loc a t ed  ata position simila r  t o  that in
E. fa ecalis 
A P H( 3 ’ )-I IIa . On the b as is of these r e su l t s , w e  suggest that  the Rv3168 med iateska nam y cin re sista nc e  in 
M. tubercu losi s
, likel y thro u gh phos p hotrans ferase  ta rge ting of  k ana my c in. 
T ube r cul osis isawo rldwi de e p i d e micdis ease , and  it has beenest imat e d t hat u p toone- th ird o f thehuman  p opulation harbo rs M ycobacter ium tu ber cu losis ,  the c aus ati ve agen t of th e  di se a s e  [ 13]. Although a co mb i n at ion of s everal an ti b iotics i snor mall y usedfort he  treatme nt oft ub er culosis ,comple te  cu reof tu b ercu losisis madediffi c ult by the exi s tence of muta n ts t ra i ns a ndthe acquisi tion of a ntib i ot icr esist ance.  A m on g  the antibiotics us ed  for t reati ng tuberculos is, aminog l y c osides a re a cl a ss of broad-sp ectru m antimicr o bial com pound s that c ontain an a mino-mod ifi edsug ar[ 1 7] .

Aminoglyco s i desha ve an a ffi nity fo r d eco din g t he  aminoacy l site o f16 SrR NAin th e  bacteri al  30 Srib osoma l  subun it, t here by  e f fec ting th e  d i s soci at io n of  am in oacyl tRNA fro m the 3 0S subuni t,r esul ti ng  in pro tein miscodin gand loss o fbac terial c ell wall  integrity [6,9]. It ha s  been s how n tha t th e major m ech anismofb acteri al res istan ce  to a minog l y co sid es  is basedo n  th e che mi calmodific ation of the antib i oti c by bacteria l e nzym e s  [ 16] .  E n zym es tha t modify aminogly cosides fo rm alarge andd ive rs e group  that i s cla s sifiedinto thre e general  c lass e s , d epending o n the ch emical gr o up of th e ant ibi otic t ha t i s mod ified; nam ely,ATP-de pe ndentphosp ho transfer ases (APH), ATPdependen t aden ylylt ran sferases(AN T ),and acety l Co Adependent  ac ety ltran sfe r ases(AAC ) .Stu d ies h aves hown seve r al  st r u ct ures of the A P H e nzyme s,i ncludi ng A PH (3’)-IIIa [1,3 , 5], APH (3’)-I Ia [ 1 0 ],APH (2’’ )- IIa [11], APH(2’ ’) - I Va [14],  a nd APH (9’) -Ia [4]. 
R ecent  studi es onthe gen o m es equenc e of  M.  tubercul osi sh ave sug ge st e d cand idat egenesrespon s ible f or the acquisiti on of a minog l yco sidere sistanc e  [12 ,16], andwe have pre viousl y re porte d the c ry stal s tru ct ure of Rv3 1 68, a put ative  aminog ly cosi dephosph otra n s feras e [7 ]. 
otransferase activity_of Rv3168_against kanamycin as a_substrate. Moreover,_docking_simulation of_kanamycin_into the Rv3168_structure suggests that_kanamycin fits well into_the substrate binding_pocket_of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position_similar_to that_in
E._faecalis
APH(3’)-IIIa._On the basis of these_results, we suggest that the_Rv3168 mediates_kanamycin resistance in
M. tuberculosis
, likely through phosphotransferase targeting_of_kanamycin.

Tuberculosis is a_worldwide epidemic disease, and it has been estimated that_up to one-third of the human_population harbors Mycobacteriumtuberculosis,_the_causative_agent of the disease_[13]. Although a combination of several_antibiotics is normally used for the_treatment of tuberculosis, complete cure of tuberculosis_is made difficult by the existence_of mutant strains and the_acquisition of_antibiotic resistance. Among the antibiotics_used for treating_tuberculosis, aminoglycosides_are a class_of broad-spectrum antimicrobial compounds that contain_an amino-modified sugar_[17].

Aminoglycosides have an affinity for decoding_the_aminoacyl site of_16S_rRNA_in the_bacterial 30S ribosomal_subunit,_thereby effecting_the_dissociation of aminoacyltRNA from the 30S_subunit,_resulting in protein miscoding and loss of_bacterial cell wall integrity_[6,9]._It has been shown_that the major mechanism of_bacterial resistance to aminoglycosides is based_on the_chemical modification_of the antibiotic by bacterial enzymes [16]. Enzymes that modify aminoglycosides_form a large and diverse group_that is classified into_three general_classes,_depending on the_chemical_group of_the antibiotic that is modified; namely, ATP-dependent_phosphotransferases (APH),_ATPdependent adenylyltransferases (ANT), and acetyl CoAdependent_acetyltransferases (AAC). Studies have_shown_several structures of the APH enzymes,_including APH (3’)-IIIa [1,3,5], APH (3’)-IIa_[10], APH (2’’)-IIa [11], APH_(2’’)-IVa_[14],_and APH (9’)-Ia [4].

Recent studies_on the genome sequence of M._tuberculosis have suggested_candidate genes responsible for the acquisition of_aminoglycoside_resistance [12,16], and we have previously_reported_the crystal structure of Rv3168, a_putative_aminoglycoside_phosphotransferase [7].