CONG NGHE SINH HQC TOAN QUOC 2013
NGHieN CCrU c a CHe XUC T A C T H O N G Q U A C A U T R U C T I N H THfe C O A FabB - ENZYME C O A VI K H U A N G A Y BAG L A L O A
Dodn Thj Ngpc Thanh Tnrdng Bai hgc Tien Giang
T6M TAT
Hi?n nay, vi khuan gSy b§nh b^c Id liiaXaniliomonas oryzarpv. oryzae (Xoo) dang hoanh hanh trSn diSn r§ng vi la m^t trong nhimg nguyfin nMn g3y ton thdt ldn cho nghi trSng lua nude 6 nirdc ta va tren the gioi. Do d6, vi?c nghien ciru co chi xiic tac cua nhung enzyme quan trgng trong yi k h u ^ nay Id buoc dSu gii^ tun ra nhihig giai phap ngan ch|n sir phat triln cua vi khu^. Nghien ciiu niy tim ra cau true tinh thS va giii thich co che xuc lic cua FabB t i Xoo. FabB, Beta-ketoacyl-synthase 1,1^ enzyme quan trgng tham gia v^o qua trinh tong hop add b6o ciia smh v|t. FabB t& Xoo dugc t^o dong, bilu liifn trong Escherichia coli. Protem tai td hgp dugc thu nh?n, tinh sach vk dugc t^o tinh th6 sau d6. Kgt qui cho fliay d u triic bac 4 ciia FabB gom 4 phSn tii. MSi phSn hi gdm 1 domain a-p-a-^a diiolase va 1 domain phy. Trung tSm hoat dgng ciia FabB c6 d?mg ong nim giiia 2 phSn tii li6n nhau vi tit khic biSt v6i trung tSm ho^t d^ng cua FabB 6 cic loii smh vit khic. Dac bi|t, hai amino acid gin voi co chit trong qui trinh xiic tic nim trong trung tam hogt d^ng rdt khic biet so vdi FabB ciia vi khuin khic. Di&i niy cho thiy ca che xuc tac ciia FabB tii Xoo rit d?c hi?u.
Tir tdioa: b?nh bjic la Kia, ciu true tinh dil, FabB, ting hgp acid beo, Xanthomonas oryzae
MdDAU
B$nh bgc Id 10a do vi khuin Xanthomonas oryzae pv. oryzae (Xoo) gUy ra lam giSm s5n lifpng lOa d nhl^u nif&c tr§n ttil gi61, toing blnh gidm 6-60% nang suit, tSng t9 1^ hat I6p (Gnanamanickam ef al.. 1999). d Vi$t Nam, tO' nam 1999- 2003 b§nh bgc Id gSy gSy hgi 108.691,4 tia di$n tich ^ n g liia; trong 66. di#n tIch bj hgi n$ng nhlt Id 156,76 ha vd di?n tich mSt trSng Id 80 ha (ttieo sd li&u th6ng kg cOa cgc Bdo v$ thi/c vgt). Do d6, cdc cong tdc chgn gi6ng khdng b$nh hay iflfc tlm ra thu6c phdng trj b$nh higu qud dd vd dang du'grc cdc nhd khoa hpc khip noi nghien ci>u. Tuy nhidn, hi$n nay vln chira d gi6ng liia khdng b$nh hay Uiu6c trj bgnh ndo thdt sg d hi$u qua.
M$t trong nhOng tii/dng nghidn ciju tlm chit khdng b$nh chuySn bi$t Id tiep c$n cau tnjc phdn tCr cOa protein tt> vi khuin gdy b$nh. Vi^c xdc djnh chi'nti xdc d u triic phdn ti> thdng qua nhilu xa tia X khi xuyen qua Unh thi cua dgi phan tfr sinh hpc dd di/pc phdt triln tir nh&ng nam 1950 (Kendrew efa/., 1958). Cac nhd khoa hgc tin rang, khi bilt du-grc clu tn)c phdn to- cOa m$t s6 enzyme quan tn;>ng cho sg sing c6n cOa vi khuin gdy b$nh, hg se nghidn ci^u vd bilt duvc co chl xiic tdc cCia chOng; tir d6 cd t h i tim ra chit irc chl chuydn bidt cho enzym nay vd xa hon la i>c chl vi khuin (Simmons et al., 2010). Trong phirang phdp ndy, bifdc khd khdn nhlt id tgo dugc tinh t h i protein cd chit Itrpng dii de Idm nhilu xg tia X vd'i d$ phdn gidi <3 A.
Ndm 2005, bO gen cua vi khuin gdy b$nh bgc Id da dupe gidi ma thdnh cdng (Lee ef al., 2005). Gen fabB Id mdt trong s6 dd. Gen fabB md hda cho enzyme FabB, Id mOt mic xlch quan trpng trong con dirdng t6ng hgp acid bdo kieu 11 chi Sirgc tlm ^ l y d vi khuin. Trong khi dd, chu5i chuyen hda acid bdo fdeu I dirge tlm thiy it eukaryote chi do mgt enzyme ddm nh$n (Rock ef al., 2002). Do si,r khdc bi^t ndy, nhOng chit irc chl FabB se ch? tdc d$ng len vi khuan md khdng gdy dnh hu'dng din ddng v^t va ngu'cri. FabB xiic tdc phdn i>ng kdo dai c^uSl acid beo vdi co chit mang acyl bio hda vd khdng bao hda. Trong khi hai enzyme cdn lai ciJa qud trlnh ndy Id FabF chl nli ddi chuSi vdi co* chit mang acyl bao hda cdn FabH chl xiic tdc phdn li'ng ban diu nli ddi acet^ cho chudi add bdo (Rock ef al., 2002). ChCnh vi ram quan trpng cOa enzyme ndy doi sg* sing cdn cua vi khuin, nd du^c cdc nhd nghidn ciru xem nhir mdt mlu chit quan trpng trong qud trinh tlm kiem chit didt khuin (Pappenberaer ef al., 2007). FabB tCr cdc lodi vi khuin gdy bdnh md phd biln nhlt la tir Ecoli da dugc tgo ddng, bilu hifn, tinh c h l vd giai quylt c l u tiiic d l u'ng dgng trong vi§c tim chit irc chl (Olsen e(
al.. 1999).
Ti> clu tnic cua rieng FabB da cd, nhdm tac gid da tgo dirge clu triic cOa phCrc hgp FabB vdi cdc ca chit de gidi thfch ro cdch lidn kit giO'a co chat vd cdc glc acid amin bong vj tri xiic tdc ciia enzyme ndy cung nhir ndu co chl xuc tdc ciia no (Olsen etal.. 2001). Nam 2011, vi§c i>ng dyng clu tnic ciia FabB tir E.co//dl tim chit irc chl da cd kit qud ban diu. Trong dd, hai hpp chit ty nhidn Id cerulenin vd thiolactomycin da du'pc bdo cdo li cd khd ndng i>c chl FabB tO
£co//{Price efa/.,2001).
Mgc dil chdng dirge thO" nghldm thanh cong trong vide iJc che mgt s6 loai vi khuin khdc nhau, nhirng gidi han v l do dgc hidu vd dd bin ciia hda chit lam gidm khd nang Ong dyng cOa chiing. Gin ddy da cd nhdm nghidn ci>u din tiJ Thyy ST bdo cdo ve chit dipt khuin di,ra trdn vi§c sdng Ipc dO- lidu cdc hpp chit hda hgc cd khd nan^ lidn kit vdi clu tnic tinh t h i cOa FabB (Pappenberger ef al., 2007). Nhdm ndy nhgn thiy aminothiazole Id mgt hgp chat tiem nang nhlt i>c chl duvc FabB trich tir E.coli, sau nhO'ng friij nghigm lidn kit di lye va thO nghipm khd ndng i>c chl hogt tinh enzyme in-vitro.
Tnang nghidn ci>u ndy, chung tdi dd thu nhgn vd tgo ddng gen fabB. bieu hign trong E. coli vd tlnh chl vol s6 lup-ng ldn prolein d l cd t h i tao dirge tmh thi. Sau khi chyp X-ray vd thu nhdn dtr lidu, clu tnic phdn tO cda FabB tir Xoo da dirpc xdc djnh vd so sdnh vdi cdc c l u tnic FabB tCr B.coli. Ddy Id bydc quan trgng ban diu cho vige tim kilm chit khdng Xoo ddc higu sau ndy.
HOI NGH! KHOA HOC CONG NGH^ SINhTR -^i^
NGUYEN u e u VA PHUOTJG PHAP Tgo ddng gen fabB, bieu h i f n vd tlnh c h l FabB
Dogn gen fedS tir chung Xoo ATCC10331 duvc khuich dgi bang cgp mil dgc higu co g i n doan md hda 6 HistidinlB'^
cho vide tinh chi sau ndy. Trinh ty moi xudi vd mdi ngypc lln luvt Id 5'-GGG CAT ATG CGT CGC GTC GTC ATC AC(
GGA AT-3' vd 5'-GG GGA TCG TCA GAC CCG GCC GAA CAC CAG G-3'. Sau dd dogn gen dirpc gdn vdo plasm*
pET tnrdc khi dirpc biln ngp vdo E.coli JM109 d l nhdn bdn. Plasmid tdi to hpp pET-fabB dirge thu nhdn vd biln n^i vdo chOng E.cof/ BL21 diing cho bieu hign protein Sy bilu hidn ciia protein FabB du-gc cdm irng bing IPTG 0,5 mMi 15°C khi mdi tnrdng nudi cly cd gid tii OD 0.6. Tir 10 lit djch nudi cly, thu lly t l bdo sau 16 gid cdm Crng. T l bdo sai dd dypc hda trong 200 ml dung djch ly giai (25 mM Tris-+ICl pH 7.5, 300 mM NaCl, 15 mM imidazola, 3 mM p mercaptoethanoi. Vide phd vo t l bao thyc hi^n bdng sdng sidu dm trong ffilu kidn Ignh. Dung djch ddng nhlt ndy di/t*
ly tam 13 000 vdng/phut ir 4°C. thu djch noi vd cho qua cdt sac ky ai lye cd gan Ni * d l tinh c h l .
Sau khi cho djch ndi vdo cot, cho dung dich chgy qua cdt mgt cdch ty nhidn. Sau dd, n>a cgt d l loai bd nhu-ng protei eo t h i tgo lidn kit khdng ddc higu vdi cpt sac k^ bang dung djch ly gidi vd dung djch n>a (gilng vdi dung djch ly gij ngogi trir 30 mM Imidazole). Bud^ ly trich protein FabB dypc thyc hidn bing cdch cho vdo cdt dung djeh chija 250 mli imidazole. Dung djch ra khdi cdt du-pc thu nhdn phdn doan mdi 10 ml. Cdc phdn dogn ndy duvc kilm tra quage awylamide d l chgn Ipc nhirng phan dogn chira FabB tinh khllt. Sau khi chgn Igc, protein tinh khilt dirge thim Mch«
trao ddi dung djch chira sao cho dung dich cudi cung cd ndng dd 25 mM Tris-HCI pH 7.5, 3 mM p-mercaptoethanol Tnrdc khi tgo tinh t h i , dung djch protein du-pc cfl dgc bing ly tdm mdng Igc cd kleh thu-dc 30 kDa, cho phdp cdc cM tgp dudi kich thuwe ndy neu cd qua mdng vd giu* Igi FabB cd kich thydc 44 kDa.
Tgo tinh t h i FabB
Phuwng phdp tgo tinh the eua protein du'pc thyc hidn dya trdn nguySn tac khuech tdn hoi (Sanderson and SItelly, 2007). Djch FabB tinh khilt vd eo dgc dupe thCr nghigm khd ndng tgo tinh the bing phuvng phdp gigt ngdi (sitting drop' hinh 1) tnrdc tidn vdi 384 dung djch mg khdc nhau. Trong mdi gilng Idn cda Oia Elisa, cd khodng ddy ldn chira 70 |il dung dieh mg vd mdt gilng nhd phia trdn chO'a 1 \ii hdn hpp bao gdm 0.5 ^1 dung djch FabB vd 0.5 pi dung dich ai^
Dung djch mg du'pc mua tir Hampton Research vd Emerald BioSystems, My. Cdc dTa ndy du-pc Ci it 12°C vd quan sS du'di kinh liip moi ngdy d l quan sidt sy xuit hi$n cda tinh thi. Sau 2 ngdy u, mdt so tinh t h i hinh que mdnh xuit hl$nA gigt treo ed dung djch mg Id 0.1 M citric acid pH 5.0 vd 2.4 M ammonium sul^te. Dung dich ndy dirpc chgn, sau dti^
mflt ddy dung dich cd pH khdc nhau vd ndng dd ammonium sulfate khde nhau d l tlm dilu kign tdt nhlt cho vigc tgo Mnh thi chat lirpng hon. Bdn cgnh dd, phuong phdp gigt treo (hanging drop- Hlnh 1) cung du'pc thyc hi$n de tdng kidi thudc tinh thi FabB. Sau nhilu nd lye, nhu'ng tinh t h i cd kfch thuwc Idn di/pc neo trong mdt vdng ddy, sau do b^
qudn bing dung djch cryo-protectant (dung djch me vd 20 % (v/v) ethylene glycol)trong dilu kign nhidt dfl Ignh ciia ni-to long.
• ^ = ^
Hinh 1 : P h v o n g phap tao tinh th4. A - h a n g i n g drop - giot treo. B-sitting d r o p - g i o t ngdi. Ngudn Wikipedia.
Thu nhan k i t qua nhieu xg tia X vd phan tfch c l u tnic
Tinh \hk da bdo qudn trong ni-to ldng dupe dua vdo hd thdng birc xg, nguon tia BL26B1 eua hd thdng Spring-8, NI Ban. Tia X dypc chilu qua tinh the, cdc nhilu xg du'pc mdy ghi nhdn Igi vd duvc chuyin sang chyong trlnh HKL-'"
xir Ij? dii ligu (Otwinowreki and Minor, 1997). DD lieu dung hpp cudi dugc dung d l phdn tfch c l u true cOa FabB phyong phdp thay t h i phdn ti> (molecular replacement). Sir dung c l u tnic ban mlu Id FabB tip E.coli (Olser a/.,1999) vd clu tnic dypc tidn doan tir trang du' lidu cCia Swiss model.
K E T Q U A V A THAO LUi\N ! Thu nhdn FabB tinh khilt
Sau khi tgo ddng, dogn gen fabB du'pc gidi trinh ty d l khang djnh plasmid tai td hgp pET-fabB dd duoc tgo thanh c&«
Td bdo mang plasmid nay dupe nudi cly vdi sd lupng Idn. Dich protein tdng s l duoc thu nhgn sau khi phd vd ti Iw bang sdng sidu dm vd ly tdm. Djch ndy dope tmh ehi qua cdt Ni-NTA vd thu duoc 6 phdn dogn chi>a protein tinh s?*
(Hinh 2 A) Pnatein trong bdng gel acrylamide cd kfch thudc 44 kDa phii hop vdi kfch thudc I;? thuylt ciia nd. Sau I**, thdm ttch va cd dgc tir 60 ml dich protein sau sdc ky, ta thu dupe 7 ml djch protein tinh sgch cd ndng dp 6.6 mg/ml. ^' nay dupe bao quan Ignh d - 80 C de chd dupe kit tinh.
Tgo tinh t h i
Sau khi sdng Ipc 384 dilu kidn, tire 384 dung dich mg khdc nhau d l tgo tinh the. quan sdt thdy mdt dilu kidn cd B * hidn nhidutinh thi nhd hinh que kim (needle) (dir lieu khdng trinh bdy). Dilu kidn nay dupe chgn d l tgo mdt ddy*"*:
: C O N G N G H £ SINH HOC T O A N Q U O C 2013
djch cd pH vd ndng dg ammonium sulfate Wide nhau. Mgt ktidc, phuvng phdp gigt beo cung duvc thyc hidn vdi nhirng dung djch mg ndy. Sau r i t nhilu thf nghiem, tinh t h i cua FabB ed chit lupng du d l thuc hign thi nghiflm bin X-ray '^SL^ ° ^ t^ gipt treo chua 2 pi protein FabB vd 2 pi dung dieh mg (0.1 M citric acid pH 5.5: 2.2 M ammonium sutfete). Tlnh t h i cd kfch thudrc 0.05x0.35x0.07 mm tfiinh 28) dupe neo trong mdt vdng nhd dudng kinh 0.3 mm; sau dd dugc nhdng vdo oyo-protectant vd Idm Ignh nhanh ttxwig nf-to ldng.
kOa Thang Faba
Hinh 2: A- prolein FabB tinh sach. B- tinh thi FabB c l u tnic phdn ti>cOa FabB
DCr li$u nhilu xg khi tia X xuydn qua tinh t h i dupe ghi nhgn vd xu ly bing chuong trlnh HKL-20OO. Kit qud eho thiy tinh t h i FabB tir Xoo cd nhdm khdng gian P4, thflng sd cda don vj c l u tnic Id a=b= 82.2 A, c = 233.2 A. SO- dyng cdc chuong trinh Scalepack vd Denzo d l xdy dyng ndn bdn dd mdt dfl electron eua phdn tir FabB (Hlnh 3A). Sau dd, su dyng phdn mim Cep4 d l phan tich clu tnic. Phin mim MOLPREP dupe dung d l so sdnh vdi cdu tnic FabB do Swiss model tgo ndn tCr bdn m l u FabB tir E.coli. Tir phdn tfch ndy, ta thu dupe c l u tnic phdn tir FabB phd hpp vdi ban dd mdt dfl electron.
c l u tnJe phdn til FabB chi>a 4 monomers. Mfii monomer gdm 14 xoan alpha vd 9 tam beta, cufln thdnh 2 domain gdm 1 domain cd d u true a-p-a-p-a vd 1 domain phy. Domain phy, gidng mpt nip d|y thioiase domain, bao gdm 5 xoln hetbcddnhdaua1.1,a1.2, 02.1,02.2 and a2.3 (hlnh 3B). Khi so sdnh cdc clu tnic FabB tir £ co//(Oslen ef a/., 1999), ta Oily domain phy ciia FabB tCr Xoo chl chCra xoln a md khdng chda philn p, dilu ndy Idm cho nd di chuyin linh hogt han trong dung djch. Oilu ddng quan tdm la domain ndy gdp phin tgo ndn trung tdm hoat dflng ciia enzyme ndn vi$c linh hogt ciia nd giiip qud trinh tilp nhdn co chit d l ddng hon.
Hlnh 3: A- khung peptide du>g>c mo phdng trong bin dd mat dg electron. B- Cau tnic tinh the ciia mgt monomer FabB.
Trung tam hogt ddng cua FabB
Dya vdo eo chl xdc tdc da dupe bdo cdo tnrdc ddy (Olsen ef al.. 1999) vd c l u tnic hidn cd, ta doan dugc tmng tdm hogt ddng cua FabB dupe tgo ra bdi sy kit hpp cCia hai monomer khdc nhau, tao thdnh mflt kdnh hgp xuydn qua vd d giOa hai phdn td' ndy. Nhu vdy, mdi monomer cua FabB cd mdt trung tdm hogt dflng nhung chf dupe hodn ^idn khi kit hgp viri mdt monomer khde (tgm ggi la MolA vd MolB). Kdnh hogt dgng ed hai diu hudng ra phia dung mdi d l tilp xue CO chit, bdn trong kdnh cd cac acid amin Hdn kit vdi eo chit trong qud trinh xuc tdc (Hlnh 4A). O l nam ro hon val trd cOa timg add amin trang trung tdm hogt dgng, tde gid cd tao tinh Oil cda phd'c hpp FabB vd eo chat dodecanoyl-CoA (DCA) vd phdn tfch c l u trdc eda nd (dO ligu khdng trinh bdy). Vide tgo tinh t h i vd thu dir lidu nhilu xg tia X cua phdc hpp ndy eung tuvng ty vdi qud trinh Idm cho FabB. Trong qud trinh nay, cP chit dupe eho thdm vdo tinh thi FabB vdi nong dfl 20 mM vd Ci trong 6 gid. Trang c l u tnic cda phirc hpp FabB-DCA, mgt kdnh cd 2 co chit gin vdo. Trong mdi diu kdnh, diu acid bdo cCia co chit DCA lien kit cflng hda tn vdi Cystein d vi tri 161 cua Moi A tgo ndn mflt ester. Gdc 0X0 ed lidn kit hydro vdi hai nguydn tir ni-ta ciia Cys 161 vd cOa Phe 390 (Hinh 4B).
Khi so sdnh bang phin mdm Pymol c l u tnic DCA-FaB tir Xoo vdi cau tnic DCA-FabB tir Eco//(Olsen ef a/.,2001), ta thiy cd sy khdc bid' v l khflng gian ciia eo chit. Dilu ndy dupe gdy ra bdi nhu'ng acid amm lidn kit vdi co chit trong
HOI NGH! KHOA HOC CONG N G H ^ SINH HC -
FabB tir Xoo khdc vdi nhirng glc tuong ung trong FabB tir E.crAi. Cy t h i nhu vj bi G»y 200 vd Pro 110 »«>"9 F ^ ^ Ecofr dirpc thay t h i bang Met 198 vd Ser 109 trong FabB cua Xoo. Hai acid amm nay cd vai trd dn djnh co chdt trong qud trinh xiic tac phdn img (Hinh 4C).
Hinh 4: A- k6nh chira hai trung tSm hogt d$ng tao n§n tii hai monomer. B- Cic acid amin tham gia vio qui trinh xiic tic. C- So Binh trung tam ho?t d^ng ciia FabB tir Xoo v i E.coli, acid amin dinh s6 l i cua FabB tir £.co/i
Co c h l xdc tdc
GiO vai trd quan trpng trong hogt dpng xCic tde eiia FabB Id Cys 161, acid amin ndy nhdn chudi aeyl tir co chit acyl-ACP thdng qua phdn dng ester. Hai acid amin His 331 vd H 296 cung ttiam gia trong qud blnh xdc tdc Wii hai vdng imida^
hudng vudng gde vdi Cys 161. Hon nua His 331 cd lidn kit hydro vdi nguydn h^SycOa Cys161 trong khi His 296 lita kit vdi Sy ciia Cys 161 thflng qua mflt phdn tir nude. Hai His nay giO' mdi tnrdng dn djnh cho phdn irng xdy ra vd c6¥|
trd hip &1V dign tO bong suflt qud trinh ester hda. Ngodi ba acid amin vira k l tham gia ehfnh vdo qud trUih xdc tdc, c^
gdc khdc cung gdp phin tham gia vdo sy quylt djnh dp ddi vd dfl bdo hda ciia co chit. Cy Oil nhu gde Giy 106, Mi 195, Met 198, Phe 199, Leu 333, Phe 390, Val 132 cOa Mot B vd Met 136 cda Moi B (Hlnh 4B). Met 198 trong FabB cflj Xoo cd khuynh hudng Idm gian kenh hogt ddng eho phdp enzyme ndy xuc tdc dupe chufii acyl ddi hon, dilu nay khfe bi§t so vdi gdc Giy 200 d FabB cua E.coli (Hlnh 40). Sy khdc biflt ndy cd j nghTa quan trgng cho nhung nghi§n ciiu tilp theo nham tlm ra chit irc chl mdi mang tinh dgc hiflu cao.
KCT LUAN
Gen ma hda cho enzyme FabB tir Xoo da dupe tgo ddng thdnh cflng. Protein FabB cOng da dupe tmh chl vdl dfl Hnh sgch cao vd ndng dfl ldn 6.6 mg/ml. Tinh Oil tip FabB cung dupe tgo thdnh tir dung djeh mg cd thdnh phin 0.1 M dtiic acid pH 5.5; 2.2 M ammonium sulfate. Clu tnic tlnh t h i eiia FabB dupe tgo ra nhd phuong phdp thay t h i phdn tirvi dupe so sdnh vdi FabB tir E.co//. Clu tnic tdng quan eiia hai phdn tir ndy khd gidng nhau nhung khde nhau d d u tnic domain phy vd vj tri xuc tdc. Td clu tnjc tinh the cua phdc hpp FabB vdi co chdt cho thiy cdi nhin cy t h i ciia qud trinh^
ester hda acyl cua e o t ^ l t (acyl-CoA) vdi gde Cys 161 cua enzyme. \ri tri xiic tdc eua enzyme cOng dvrgc so sdnhi""
FabB tir E.coli cho thiy ed sy khde biflt d vi tri Met 198 vd Ser 109. Hal gdc nay Igi tham gia vao qud trinh quylt djnh' ddi vd dfl bdo hda oia co chit. Nhu thi, co chl xuc tdc cda FabB da dupe minh hpa thdng qua c l u trdc tinh thi o FabB vd FabB-DCA. Ddy Id budc quan trgng trong hudng nghidn cdu cac hpp chit cd khd ndng irng chl FabB tirfh irc chl vi khuan gdy bgc Id Ida.
LM cftm Ofn: Nghien ciiu duac iht/c hien v&i sif ko trp vi ky thu^l va tai chinh cua giao sir Lin-Wao Kang va tien siJIn-Kwang Kim, phiiigM nghifm toan cdu cua trvang Dai hoc Konhik, Han quoc. Cong vifc & hf thong bin: xg duac ho trp bai cac nha khoa hpc 6 Sprtng-8, Nhft Bin. ' 1
T A I L I $ U T H A M K H A O
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Kim JH, Jung CH. Koh NH. Seo JS & Go SJ (2005) The genome sequence of Xantfjomonas ofyzae pathovar oryzae KACCIoasi.W bacterial blight pathogen of nee Nucleic Acids Res 33(2): 577-86.
Olsen JG, Kadzioia A, Wettstein-Knowles VP, Siggaard-Andersen M, Undquist Y & Larsen S (1999) The X-ray ciystal stmclureof b * ketoacyl [acyl earner prolein] synthase I FEBS Lett 460(1): 46-52.
Olsen JG,Kadziola A,von Wettstein-Knowles P,Siggaard-ZVndersen M KLarsen S (2001) Structures of beta4cetoacyl'ac)^ can*
protein synthase I complexed with fatty acids elucidate its catalytic machinery. Stnjcture 9(3): 233-43.
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: .QC C 6 N G NGHf SINH HQC TOAN QUOC 2013
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STUDY OF CATALYTIC MECHANISM OF FABB FROM XANTHOMONAS ORYZAE PV. ORYZAE VIA CRYSTAL STRUCTURE
D o a n , T h i N g o c T h a n h
Agricultural Engineering and Food Technology Department, Tien Giang University
Nowadays, Xanthomonas oryzae pv. oryzae (Xoo) causing bacterial blight disease of rice has huge effect on rice destruction in rice growing countries including Viet Nam. Therefore, study of catalytic nteclianism of vital enzymes from the bacteria is considered as a first important step to develop new agents against bacterial ^owdi.
This study solved crystal structure and explained catalytic meclianism of FabB fiom Xoo. The enzyme called Bela-ketoacyl-synthase lis an important enzyme in fetty acid synthesis pathway of tiacteria. FabB gen from Xoo was cloned, tlie protein was expressed in Escherichia coli. Then the recombinant protein was purified and crystallized. The solved structure contains four monomers in an asymmetric unit wliile it exists in solution under dimer form as runmng native polyacrylamide gel. One subunit contains 14 a- helices and 9 P-sheets, folds to form an a-^-a-^a ttiiolase structure and a subdomain. The active site of FabB is created at the intei&ce of two different monomers. The channel-shaped active site in the interfiice is different from Icnown FabB active site of other bacteria. Especially, two substrate binding residues in FabB are also different from the courteipart in other known FabB structures.
This result showed specificity of catalytic mechanism of FabB fiom Xoo, which is an inqrartant step to find novel inhibiting agent Key words: bacterial blight, crystal stmcture, FabB, tatty acid synfliesis, Xanthomonas oryzae.