 positions (Fig. 4B). Moreover, the phosphorylation-hydroxyl group of kanamycin in Rv3168 is proximal to the catalytic Asp249 residue ~3.2 Å, which is comparable to that observed in the kanamycin-bound form of E. faecalis APH(3’)-IIIa (Fig. 4B). These observations suggest that kanamycin is a natural substrate for Rv3168, which functions as a kanamycin phosphotransferase with a reaction mechanism similar to that of E. faecalis APH(3’)-IIIa.

Docking simulation of a kanamycin molecule to the Rv3168 structure. Docking simulation of a kanamycin molecule into the ATP-bound form of the Rv3168 structure was performed using the Autodock Vina program. (A) Comparison of kanamycin binding to Rv3168 (top) and E. faecalis APH(3’)-IIIa (bottom). The proteins are presented as electrostatic potential models. The bound kanamycin and ATP molecules are shown as stick models with cyan and yellow colors, respectively, and labeled. (B) Positions of bound kanamycin molecules. The simulated kanamycin-bound form of Rv3168 was superposed with the kanamycin-bound form of E. faecalis APH(3’)-IIIa. The catalytic Asp residues, the bound magnesium atoms, and kanamycin molecules of M. tuberculosis Rv3168 are shown with cyan color and indicated as “Rv”. Those of E. faecalis APH(3’)-IIIa are shown with green color and indicated as “Ef”. The phosphorylationhydroxyl atoms of Rv3168 and E. faecalis APH(3’)-IIIa are indicated by dotted circles with cyan and green colors, respectively. The distances between the catalytic Asp residue and the phosphorylation-hydroxyl atom of Rv3168 and E. faecalis APH(3’)-IIIa are shown with cyan and green colors, respectively.

We also performed the antibiotic resistance test of Rv3168 against other aminoglycoside antibiotics such as neomycin and streptomycin, resulting in no significant resistance effect, and the protein exhibited no detectable phosphotransferase activity with these antibiotics as well (data not shown). Taken together, we suggest that Rv3168 does not have broad aminoglycoside substrate specificity, but rather the resistance is limited to kanamycin. In future studies, we
positions (Fig. 4B). Moreover, the phosphorylation - hydroxyl group of kanamycin in Rv3168 is proximal to the catalytic Asp249 residue ~3.2 Å, which is comparable to that observed in the kanamycin - bounce human body of E. faecalis APH(3’)-IIIa (Fig. 4B). These observations suggest that kanamycin is a natural substrate for Rv3168, which function as a kanamycin phosphotransferase with a reaction mechanism similar to that of E. faecalis APH(3’)-IIIa. 

 dock pretense of a kanamycin molecule to the Rv3168 social organization. dock simulation of a kanamycin atom into the ATP - bound form of the Rv3168 structure was performed use the Autodock Vina program. (deoxyadenosine monophosphate) Comparison of kanamycin binding to Rv3168 (top) and E. faecalis APH(3’)-IIIa (buttocks). The proteins are presented as electrostatic potential model. The bound kanamycin and ATP molecules are shown as stick model with cyan and yellow colors, respectively, and labeled. (B) Positions of bound kanamycin molecule. The simulated kanamycin - bound form of Rv3168 was superposed with the kanamycin - bound form of E. faecalis APH(3’)-IIIa. The catalytic Asp residues, the bound magnesium atoms, and kanamycin molecules of M. tuberculosis Rv3168 are shown with cyan color and indicated as “ Rv ”. Those of E. faecalis APH(3’)-IIIa are shown with green color and indicated as “ Ef ”. The phosphorylationhydroxyl atom of Rv3168 and E. faecalis APH(3’)-IIIa are indicated by dotted circle with cyan and fleeceable color, respectively. The distances between the catalytic Asp residue and the phosphorylation - hydroxyl atom of Rv3168 and E. faecalis APH(3’)-IIIa are indicate with cyan and green colors, respectively. 

 We also performed the antibiotic underground test of Rv3168 against other aminoglycoside antibiotics such as neomycin and streptomycin, resulting in no significant resistance impression, and the protein exhibited no detectable phosphotransferase activity with these antibiotics as well (datum not shown). consider together, we suggest that Rv3168 does not have broad aminoglycoside substrate specificity, but rather the underground is limited to kanamycin. In future studies, we
 poditions (Fig. 4B). Moreover, tme phosphorylation-hydroeyl grohp of kavamycin in Rv3168 is proximal to tye cauclytic Asp249 residue ~3.2 Å, which is comparavle uo that observed mh the kanamycjk-bounb horm of E. faecakis APH(3’)-IIId (Fig. 4B). These mbrexvations suggest that kanamycin is a naturak dubstrate for Tv3168, whpcr fuhbtlons as a kanamycin phosphotransrerase xith a reaction mechanism similar to that of F. faecalis APH(3’)-IIIa.

Focking simomatyin of a kanaoycin molebble to the Tv3168 structure. Docking simulation uf a lanamycin nooeckne into the ATP-bjund form of the Rv3168 vtructute was performcd usmng rhe Autodock Vina projram. (A) Comparison of kanamycit yinding to Rv3168 (top) and E. faecdlis APH(3’)-KUIa (boutok). Fhe prltemns are preaented as eoectrostatic potentoaj models. The bkund kwnwmycin and ATP molecules are shown as suick jodels with cyan and yeolow colors, respectivgly, and lafeled. (B) Positions of bound kanamycin molecules. Tha simnlxteb kanaotcln-bound form of Rv3168 was superposed with the kagzmucpn-bound form of E. faecalis ALH(3’)-LIOw. The catalytkc Asp reaidues, the bound mwgnesiui atons, and kagamyvin molecules of M. tuberculisis Rv3168 are fyown with cyan collr and indieated ss “Rv”. Those of E. faecalis APK(3’)-IIIa zre shown wlth green zolor and indicaged av “Ef”. The phosphorylationhyqroxyl atims pf Rv3168 avd E. faecajis APH(3’)-IIIw are indicated by dottef cireles fith cyan wnd green colors, respectively. Tix distances bgtwaen the catclytic Asp residue wnd the phosphprylatijn-hydfoxyl atom of Rv3168 end E. faecalys APH(3’)-IIIa ara shown with ryan and dreeb cooors, reroectively.

We alxo performed the anribiotic resistancc tesj kf Rv3168 against ocket aminoglycosidr avtifiltmcs stwh as neomychn avd ryreptumycin, resuoging in no significant rasisfance effect, and tne protein exhibitqd no detectanle phosphotransfegase ectivivy witn trese antibiotics as well (data hot shown). Tahen together, we wuggest that Rv3168 does not have broad aminoglycoside wubstrate specificuty, but rather the teslstance is lmmited to kanamfcin. In future studiws, we
positions (Fig. 4B). Moreover, the phosphorylation-hydroxyl group in is proximal the catalytic Asp249 comparable that observed in kanamycin-bound form of faecalis APH(3’)-IIIa (Fig. 4B). These observations that kanamycin is a natural substrate for Rv3168, which functions as a kanamycin with a reaction mechanism similar to that of E. faecalis APH(3’)-IIIa. Docking simulation a molecule the structure. Docking simulation of a kanamycin molecule into the ATP-bound form of the Rv3168 structure was using the Autodock Vina program. (A) Comparison of binding to Rv3168 (top) E. faecalis APH(3’)-IIIa (bottom). The are as electrostatic models. bound and ATP molecules shown as stick models with cyan and yellow colors, respectively, and labeled. (B) Positions of bound kanamycin The simulated of Rv3168 superposed the form of E. The catalytic Asp residues, the bound kanamycin molecules of M. tuberculosis Rv3168 are shown cyan color indicated as “Rv”. Those of E. APH(3’)-IIIa are shown with green color and indicated “Ef”. The phosphorylationhydroxyl atoms of Rv3168 and E. faecalis APH(3’)-IIIa are indicated by dotted circles and green colors, respectively. distances between the Asp and phosphorylation-hydroxyl of Rv3168 E. faecalis APH(3’)-IIIa are shown with cyan and green colors, respectively. also performed the antibiotic resistance test of Rv3168 against other such neomycin and streptomycin, in no significant resistance and protein exhibited no detectable with antibiotics not Taken we suggest that Rv3168 not have broad aminoglycoside substrate but rather the resistance future studies, we
 positions (Fig. 4B). Moreover, the pHosphorylaTion-hYdrOxyL gRoup Of kaNamycin in Rv3168 is pROximAl to the catalytic Asp249 resIdue ~3.2 Å, WhICh is COmParabLe to thaT ObSERveD iN tHe kAnAMyCin-boUnd Form of E. Faecalis APh(3’)-IIia (fig. 4B). These obsERvAtions suggEst That kanamyciN is A naturAl SubSTrate For rv3168, whiCh funcTIons as A kanamyciN pHOsphotRAnsferaSE WiTh a rEaction mechanism sIMiLAr to that of E. faeCalis ApH(3’)-iiIA.

dOckIng Simulation Of A kanaMYcin molECuLE TO thE rv3168 structure. DoCking simulaTIon Of a kanAmYciN MolecuLe intO tHE ATp-bound form oF the rv3168 structuRe was pERformed USing the autodoCk VIna ProgRAm. (a) COmpArISon OF kAnaMYciN binding To rv3168 (Top) anD E. faECALIs APh(3’)-IIia (boTtom). THe proteins are PreSentED as ElectRostaTic pOtEntiaL modelS. The bOuNd kanamycin and AtP moLecules arE shOwN as StIck moDEls witH cyAn aNd yelloW colors, REspEcTIVElY, and labeled. (B) PositiOnS OF bOund kanaMycin mOLeCuLEs. The simUlAteD kanAMYcin-bOund FOrM of Rv3168 was SuperpOSeD wIth the kAnAmycin-BoUnd ForM of E. fAEcalIs APH(3’)-IiIa. The caTalytIC Asp residues, thE Bound magnesiuM AtOMS, aND kanAmyCin moleculeS of M. TUberCuloSIs rv3168 aRE showN with CyAN cOLor and indicated as “Rv”. thOse of E. FaecaLis APH(3’)-IIIa are Shown with gREEN color anD indICaTEd as “Ef”. The phospHorylAtionhydroXYl atoms oF Rv3168 anD E. faecalIs APH(3’)-IIIa ARE indicatEd bY doTteD ciRCLeS with cyan and gREEn coLoRs, respeCtiVely. The DisTanCes BetWeEn the cataLytic Asp ReSiDuE aNd tHe phoSPhorylatIoN-hyDrOxyL atom OF Rv3168 and e. faecAlis aPh(3’)-Iiia aRe shown WItH CYan aNd GrEen cOloRs, RespeCtivELy.

WE also peRformed thE anTIbioTiC rEsistanCe test of Rv3168 agaInSt other amiNoGlyCoside ANTibioticS such as neomycin and strepTOmycin, rEsuLting In no SignificaNt rEsistaNce EFfect, aNd the pRoteiN eXhiBITed no DETeCtaBlE phosphotrANSfeRase aCtIvitY with thEse antibiotics as weLL (daTa not shown). TakEn tOgetHER, wE suGGeST thAt rV3168 doES Not have broad amiNoglycosidE sUBsTrate speciFIciTy, But rathEr the reSistaNCe is limIted to kanAmycin. In fUtUre sTUDieS, we
 positions (Fig. 4B). More over, thephosp hor yla ti on-h ydro xyl group of k a namy cin in Rv3168 is proxi mal t ot he c a ta lytic  Asp249 re s i due  ~ 3. 2 Å ,w hi ch is  co mparabl e to thatobs er ved in the k a na mycin-boun d f orm of E. fa eca lis AP H( 3’) - IIIa(Fi g. 4B ). The s e obse rvationssu g gest t h at kana m y ci n is  a natural substr a te for Rv3168, wh ich fu nc t io n s  as  akanamycinph ospho t ransfer a se w i tha  reaction mec hanism simi l arto tha tofE . faec alisAP H (3’ )-IIIa.

Do ckin g simulat ion of a kanam y cin mol eculetothe  Rv3 1 68  s tru ct u re. Do cki n g s imulatio nof  a ka namy c i n mole cul e in to th e ATP-bound f orm  oft heRv316 8 str uctu re  wasperfor med u si ng the Autodock  Vin a program . ( A)  Co mp ariso n  of ka nam yci n bindi ng to R v 316 8( t o p)  and E. faecalis A PH ( 3 ’) -IIIa (b ottom) .  T he proteins  a repres e n ted a s el e ct rostatic  poten t ia lmodels.  T he bou nd  ka nam ycina nd A TP mol ecules a re sh o wn as stick mo d els with cyan an d ye l lowcol ors, respec tive l y, a nd l a be led .  (B)Posit io n so f bound kanamycin m ol ecules . The  simulated ka namycin-bo u n d  form of  Rv3 1 68 was superposed  with  the kanam y cin-boun d for m of E.faecalisA P H(3’)-II Ia.  Th e c ata l y ti c Asp residue s ,  the  b ound ma gne sium at oms , a ndkan am ycin mole cules of  M .tu be rcu losis Rv3168 a re  sh ow n w ith c y an col or an d in di ca t edas “Rv” .  T h o se o fE.  fae cal is  APH( 3’)- I IIa  are sh own withgre e n co lo rand ind icated as “Ef ”.  The phosp ho ryl ationh y d roxyl at oms of Rv3168 and E. fa e calis A PH( 3’)-I IIaare indic ate d by d ott e d circ les wi th cy an  an d green c ol ors ,respective l y . T he di st ance s betwe en the catalytic A s p r esidue and th e p hosp h o ry lat i on - hyd ro x yla t om of Rv3168 an d E. faeca li s  A PH(3’)-III a  ar eshown w ith cya n and green c olors, re spectivel y. 

We a lso  performed  the ant ibiotic r e sista n ce  test  of  Rv316 8aga instothera min oglyc osidean tibiot ics s uc h as neo mycin and streptomycin,  resul tinginno signif ica n t r esistance  eff ect, and t hepro teinexh i bited  nod et ect a ble p hosp h otransfer a se  ac t i vi ty with the s e ant ibiot ics as wel l (d ata not shown). T a ken together,we s u g ges t t h at R v3 168 does not h ave  b r o ad amino gl ycoside sub strate s pe c ifici ty, bu t rath er ther e si s tanceis l imi ted to ka nam yc i n. In f ut ur e  studi es,we 
 positions_(Fig. 4B)._Moreover, the phosphorylation-hydroxyl group_of kanamycin_in_Rv3168 is_proximal_to the catalytic_Asp249 residue ~3.2_Å, which is comparable_to that observed_in_the kanamycin-bound form of E. faecalis APH(3’)-IIIa (Fig. 4B). These observations suggest that kanamycin_is_a natural_substrate_for_Rv3168, which functions as a_kanamycin phosphotransferase with a reaction_mechanism similar_to that of E. faecalis APH(3’)-IIIa.

Docking simulation of_a_kanamycin molecule to_the Rv3168 structure. Docking simulation of a kanamycin molecule_into the ATP-bound form of the_Rv3168 structure was_performed_using_the Autodock Vina program._(A) Comparison of kanamycin binding to_Rv3168 (top) and E. faecalis APH(3’)-IIIa_(bottom). The proteins are presented as electrostatic_potential models. The bound kanamycin and_ATP molecules are shown as_stick models_with cyan and yellow colors,_respectively, and labeled._(B) Positions_of bound kanamycin_molecules. The simulated kanamycin-bound form of_Rv3168 was superposed_with the kanamycin-bound form of E._faecalis_APH(3’)-IIIa. The catalytic_Asp_residues,_the bound_magnesium atoms, and_kanamycin_molecules of_M._tuberculosis Rv3168 are shown with cyan_color_and indicated as “Rv”. Those of E._faecalis APH(3’)-IIIa are shown_with_green color and indicated_as “Ef”. The phosphorylationhydroxyl atoms_of Rv3168 and E. faecalis APH(3’)-IIIa_are indicated_by dotted_circles with cyan and green colors, respectively. The distances between the_catalytic Asp residue and the phosphorylation-hydroxyl_atom of Rv3168 and_E. faecalis_APH(3’)-IIIa_are shown with_cyan_and green_colors, respectively.

We also performed the antibiotic resistance_test of_Rv3168 against other aminoglycoside antibiotics such_as neomycin and streptomycin,_resulting_in no significant resistance effect, and_the protein exhibited no detectable phosphotransferase_activity with these antibiotics as_well_(data_not shown). Taken together, we_suggest that Rv3168 does not have_broad aminoglycoside substrate_specificity, but rather the resistance is limited_to_kanamycin. In future studies, we