velikost textu

Studium biosyntetické dráhy antibiotika linkomycinu

Upozornění: Informace získané z popisných dat či souborů uložených v Repozitáři závěrečných prací nemohou být použity k výdělečným účelům nebo vydávány za studijní, vědeckou nebo jinou tvůrčí činnost jiné osoby než autora.
Název:
Studium biosyntetické dráhy antibiotika linkomycinu
Název v angličtině:
The study of the biosynthetic pathway of the antibiotic lincomycin
Typ:
Disertační práce
Autor:
Mgr. Jitka Novotná, Ph.D.
Školitel:
prof. RNDr. Jaroslav Spížek, DrSc.
Oponenti:
RNDr. Jaroslav Weiser, CSc.
Ing. Juraj Gašparík, CSc.
Id práce:
112684
Fakulta:
Přírodovědecká fakulta (PřF)
Pracoviště:
Katedra genetiky a mikrobiologie (31-140)
Program studia:
Mikrobiologie (P1510)
Obor studia:
-
Přidělovaný titul:
Ph.D.
Datum obhajoby:
26. 9. 2008
Výsledek obhajoby:
Prospěl/a
Informace o neveřejnosti:
Příloha práce byla vyloučena ze zveřejnění.
Jazyk práce:
Čeština
Abstrakt:
_________________________________________________________________________ SOUHRN Závěrem lze shrnout, že v rámci disertační práce byly: 1) identifikován substrát (tyrosin) a produkt (DOPA) první reakce v biosyntéze PPL. Katalýza této reakce byla přiřazena výhradně proteinovému produktu genu lmbB2, čímž bylo zároveň potvrzeno, že lmbB2 není pseudogen, ale skutečně funkčním genem linkomycinového biosyntetického shluku genů, který katalyzuje hydroxylaci tyrosinu na DOPA a to bez přispění proteinu LmbB1. Protein LmbB2 byl charakterizován jako hemoprotein, který využívá k hydroxylaci aromatického jádra tyrosinu jako zdroj redoxních ekvivalentů BH4. Jde tedy o enzymovou aktivitu, jejíž mechanizmus nebyl dosud popsán. 2) potvrzen substrát (DOPA) a identifikován produkt kyselina 4-(3-karboxy-3oxo- propenyl)-2,3-dihydro-1H-pyrrol-2-karboxylová) druhé reakce v biosyntéze PPL. Katalýza této reakce byla přiřazena proteinovému produktu genu lmbB1. Protein LmbB1 byl potvrzen jako 2,3-extradiol dioxygenasa štěpící aromatické jádro tyrosinu. Na základě analýzy experimentů s třemi analogickými enzymovými aktivitami byla vyslovena hypotéza o přímé účasti proteinu LmbB1 v intramolekulární cyklizaci 2,3-secoDOPA. 3) nalezeny vzájemné interakce mezi kandidátními proteiny na podjednotky NDL- syntetasy. Na základě kvalitativní analýzy protein-proteinových interakcí metodou dvouhybridového systému a srovnávací analýzy sekvencí testovaných proteinů lze říci, že proteiny kodované geny lmbD, lmbE lmbF a lmbN lze velmi pravděpodobně považovat za významné kandidáty na podjednotky NDL- syntetázy, protože LmbD vykazuje interakci s proteinem LmbC, jehož souvislost s kondenzační reakcí byla prokázána experimentálně a zároveň vstupuje do sítě interakcí tvořených proteiny LmbE, LmbF a LmbN. Protein LmbE by mohl hrát roli centrální katalytické podjednotky, LmbC protein roli proteinu aktivujícího PPL a protein LmbN roli ACP. Celý komplex by mohl být řízen proteiny LmbQ a LmbIH. Role LmbF a LmbD je za současného stavu znalostí těžko předpověditelná. - 100 - __
Abstract v angličtině:
SUMMARY 1. L.3'4-Dihydroxyphenyl a|anine.extraďo| cleavage is followed by intra-molecular cyclization in lincomycin biosynthesis. The aim of the work was to assignfunction to the proteincoded for by an lmbBl gene, characterize betterand confirm the assumption it that 2,3-extradiol fission of the DOPA aromaticring is the actualreactioninvolved in the metabolicpathwayleadingto lincomycinsynthesis. HrN-) a.Ť:+*.i'} fooH OH ,,"1 cooH ,,"4 cooH /t{3Íboxy+ox}prcponý}2'34|hyÚ} 1/í tyÍo8|n 2'3.6ÓcoDoPA pyÍrolc2íboxy|lo rc|d Fig' 1 |nitia|steps of the amino acid subpathway oÍthe |incomycin biosynthesis The results of the feeding experiments with labeled intermediates and subsequent NMR analysis(BRAHME et al., 1984), beared witnessof the fact thatthe aminoacid sub- pathwayof the líncomycin includes2,3-extradiol biosynthesis cleavage DoPA (Fig. 1). of Neusser and coworkers (NEUSSER et al., 1998) showed that LmbBl catalyzes conversion DOPA to an unspecified of yellow compound. It appearedinapplicableto isolate the LmbBl reactionproductdirectly from in y,itroreacÍion catalyzedby the purified LmbBl partly as LmbBl lost most of its activity duringdialysis.most probablydue to oxidationof the ferrousion proposedas a cofactor, and partly due to the fact thatmany diÍ.ferent DoPA oxidationproductswere producedin the system. Instead,a system simrlar to that applied Íbr identificationof phenazine biosynthesis intermediates (MCDONALD er al., 2001)was used.The in vivo systemwas moreefficientand also produced fewerDOPA oxidationproducts. The LmbB I reactionproductisolatedfrom the mediumin the in uilr.; systemhad nrigration characteristics identicalwith thoseof the compoundisolatedfrom the puriÍied LmbB 1 directed reactionuponCE sepaÍation. Combinationof the high separation efficiencyand detection sensitivity togetherwith a soti ionization technique (CE-ESl-MS) used for the compound analysis allowed determination the LmbBl reactionproduct-smolecularmass and also its fragmentation of pattern (Fig. 2). The puriÍiedLmbB l reactionproductisolatedfiorn the ill vilro systemand the compoundisolatedfiom that of llt vjvr;showedthe same mígration time and providedan iclenticalpseudomolecular [M H]---> nlz 210. The fragments the [M-H]--rm/z ion of 210 were interpretedas products of decarboxylation([M-H] ----) nt]z 166), decarboxylation and dehydration([M-H]+ rnlz 148) and both decarboxylation and decarbonylation (tM-Hl ---+ m/z 138). The mass spectrawere in agreement with thoseexpectedfor 4-(3-carboxy-3-oxo- propenyl)-2,3-dihydro-lH-pynole-2-carboxylic predictedas an intermediate tbe acid in propylproline biosynthesis(BRAHME et al., 1984). LmbBl contains a dioxygenasesignature(NEUSSER et al., 1998) and shows domainof the VOC (vicinal oxygenchelate) significantsimilarityto conserved statistically superfamily(superfamilyínvolvingamong other type I extradioldioxygenases cleaving aromatic rings)(VAILLANCOURT et aL. 2006).It can thusbe concludedthatLmbB I is a , 2,3-extradiol dioxygenase cleavingthe DOPA aromatic ring. (llltcorcol ! 210 r50 m/. m/z Fig.2.cE-Mso|thé product Íragmentation reaction LmbB1 and pattern. 2,3-extradiolfission of the DOPA aromatic ring is proposed the biosynthesis in of muscaflavin(BUGG a WINFIELD, 1998), stizolobinic acid (SAITO a KOMAMINE, 1978) and pynolo[1,4]-benzodiazepine antibiotics (HURLEY et a/., 19'79) and lincomycins (BRAHME et al., 1984). Although the reactionsyield the same primary product, 2,3-secoDOPA, the next cyclization step produces different heterocyclic substances:muscaflavin.stizolobinicacid or 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro- lH-pynole-2-carboxylic acid (Fig. 3). While the 2,3-extradiol fission reactionis assumed to be enzymaticallycatalyzedin extensoin S. hasjoo (SAITO a KOMAMINts, 1978),a purely spontaneous 2,3-seC()DoPA cyclization leading to muscaflavinis expectedin Á' muscaria.However.the formationof muscaflavinfrom the isolated2.3-secoDOPA was not complementary decay of 2,3-.secoDOPA to and an importantpart of 2.3-secoDOPA disappeared unknown products (TERRADAS a WYLER, l99l). Consequently, to the evidenceof the third alternative the 2,3-secoDoPA cyclizationleadsto a hypothesís of of the employmentof the discussedenzymesin the primary productcyclization.Extensive studies thesereactíons comoÍrrative of could be illumínatine. 2,3-3#oOOPA Ý@ r"' .\ _"C^ : *-, ",.\ .,.1 ".") - í\ *^rr< - "- L l .Ý1 *1it-. V imosrtsction.íoduct I *l \ '^ oo"o \ I lyros|né (^\* \ l i * .5 'u"""rt"utn l.* ..1 sll2oloblnic ecld ""{}o.,."".-o,o Fig. 3 ooPA aromatic řing cleavage 1 fo||owed by intřamo|ecu|arcyclization oÍ . L lhe ploduct occuÍs inbiosynthesis oÍ "\ ...]\-\...^ -^ 1.'(.- |incomycin and otheÍ biosynthetica||y related sekundary metabolites' srrrorobrc acrd xralamic acrd -6- 2. LmbB2. a unique heme.containing tetrahydrobiopterin (BHa) dependent nzyme hydroxylating tyrosine aromatic ring in lincomycin biosynthesis (publication in preparation). The ainr of the work was to assign Íunction the LnbB2 protein,characterize to it betterand confirm an assumption thattyrosinehydroxylation yielding DopA is the actual reactioninvolvedin the metabolic pathwayleadingIincornycin. The results of the feeding experiments with labeled intermediates and subsequent NMR analysis(BRAHME et al., 1984),showedthar rhe amino acid sub-pathway the of lincomycin biosynthesis includeshydroxylation tyrosinearomaticring to yield DOPA oť (Fig. l)' however.the reactionhas neitherbeenassÍgned proteinproductof any geneoť to the lincomycin clusterin experimentsirtvitro, nor the respective proteinhas so far been characterized. Neusserand coworkersshowedthat if co-expressed E.<:oli in the InbBI a lnbB2 genesensureconversionof tyrosineor DopA to an unspecified yellow compound (NEUSSER et al., 1998).Tyrosine hydroxylationactrvityhas not been demonstrated rn viÍro and the interpretation thesedata was that the LmbB2 proteineither alone or tn of accordwith LmbB I can hydroxylate tyrosinearomaticring. the The LmbB2 protein was overproducedtn E. coli at a lowered post-induction temperature purified by affinity chromatography its active form. Reactionproduct and in of thepurifiedLmbB2 showingmigrationcharacteristics identicalwith theDOPA standard was analyzed by HR-MS. Irs m/z was identical with rhar of DOpA standard(Fig 4). consequently,LmbB2 is a monooxygenase catalyzingincorporation the oxygen atom of into the tyrosinearomatic ring. Fig. 4 HR.MS ana|ysis of HPLC puriÍied Lmb82 reactíonpÍoduct. 2 1: ; lÍ - 19 ls ís '.7 1e8 lB 2m 20' il2 LmbB2 exhibits two pH optima of 8 and 9 and a temperature optimum at 55oC, however, stabilitydecreases its afrera I O-minincubation 25.C and higher. at LmbB2 absorprion specrrum had a maximumar 403 nm (Soretband)(Fig. 5) and its Raman spectrumwas alnrostidenticalwith that oť protoporphyrin (Fig. 6) indicating IX that type b heme is presentin the LmbB2 molecule. LmbB2 is thus a heme-containing tyrosrnemonooxygenase. There are many b-type heme oxygenases.members of the cytochromeP450 superfamily,hirdroxylatingaromatic rings in bacteria(ULLRICH a HOFRICHTER, 2007).surprisingly,LmbB2 does nor sharetheir typical fearurer.e. rhe I absorption maximum at 4-50nm in their CO-reduceddifferentialspectra.causedby the presence cysteineligandof theirhemeFe (ULLRICH a HOFRICHTER, 2007). of I ail >. ll o ro 7 o C .o na E-f"=-"^"J^L/- o !04 É 03 (, tr D2 OJ 0 380 430 480 530 580 630 680 wawe|enoth Ínml Fig.5. (A) Absorption spectru oxidized ot (Íull Fig. 6 su.-Íace.enhanced resonance Raman Iine) and recluced (dotted line) form of spectra ot LmbB2 (e),protoporphyrin lX LnbB2, (B) Beduced Co4ifferential (b) a hemetoporphyrin (c). spectrum ot LmbB2. Monooxygenation tyrosinearomaticring requiresdonationof redox equivalents. of No detectable amountof dopaquinone was found in the LmbB2 reactionreflecting role the of DOPA as a donorfound in tyrosinases. Therefore, severalreducedcofactors weretested- Of them NAD(P)H ratherdecreased LmbB2 activity.Nevertheless, the BH+ addedto the LmbB2 reactionmixtureincreased activity almosttwice. i.e. BHr can reducethe heme its in the enzyme.In addition'the LmbB2 reactionpeďormed with BH+ as a co.substrate followed Michaelis-Mentenkinetics in preliminaryexperiments. These findings suggest thatBH4 could be a physiological reducingagentof the LmbB2 heme. oniy a small numberof enzymes'includingtyrosinehydroxylases, utilize BH4 can as a cofactorand the one and only hen-roprotein known to require BHl is NOS, a now flavo-hemoprotein which catalyzesa two-stepNADPH- and O2-dependent oxidation of arginineto generate and citrulline(STUEHR et al.,2005). Surprisingly. flavin was NO no found in the LmbB2 molecule.Nevertheless. experimental an evidencefor the anaerobic reductionof a group of structurallydiverse hemoproteins tetrahydrobiopterins by from Fe(lII) to the corresponding Fe(ll) statehas been reported(CAPEILLERE-BLANDIN er al., 2005) confirming that the one-electron transferfrom tetrahydrobiopterins FeIII- to porphyrins (obligateone-electron acceptor(WALSH, 1980)is a generalreaction. The abovementioned characteristics in accordance are with the fact.thatalthough the LmbB2 protein catalyzes a monooxygenation tyrosine aromatic ring, it shares no of significanthomology with any of the protein family known to catalyze the reaction. Moreover,with one exception (HU et al., 2007)LmbB2 did not show significantsimilarity to any known protein.This uniqueness the LmbB2 amino acid sequence. of surprisingly. does not contÍast with the fact that LmbB2 houses such a frequentprostheticgroup as heme b. Heme b have been found in over 20 differentfolds (SCHNEIDER et al.,2007). Consequently,LmbB2 is a unique heme-containing BHa-dependent monooxygenase hydroxylating tyrosinearomatic ring. -8- 3. Search for subunits of NDl-synthetase, the key enzyme of the lincomycin biosynthetic pathway (data for publication in preparation). The aim of the work was to find interactions amongthe proteincandidatesfor of subunits the NDl-synthetase, key enzymeof lincomycinbiosynthesis the (Fig. 7). tyrosane 9rwose ( G I Ý I Ý o GI PPL MTL o Fig' 7 scheme oí the |incomyc.n I o y' NDL-sYntheBe C' biosynthesis. o o o N.dmthy||incomycin o E I lincomycin NDl-synthetasecatalyzíng condensation the key intermediates the lincomycin of of biosynthesis (PPL a MTL; (CHUNG er al.,1997))is a complexof readilydissociable non- identicalsubuni(s which have not been identified nou'.Sequence by analysis,inactivation. Western blot and activity experimentshelped to choose protein candidatesof NDL- synthetase subunits related or proteins (LmbC,D.E,F,lH.Qand T). Except for LmbC, the function of none of the candidate proteins has been demonstrated experimentally. LmbC catalyzesPPL activation,which probably precedes the condensation Íeaction itself. The knowledgeof interactions the candidateprot'eins of with LmbC can bring very valuableinformation. addirion,since proreinsusuuily hau. In about 5 interaction partners(WILDOVA a RUMLOVA, 2008) ir can be expectedthat LmbC would be a partof the netof interactions the NDl-synthetasecomplex. in Reciprocal interactions among the candidate proteinswere testedby means of the yeasttwo.hybridsystemin pairwiseaÍrangement. Full-lengthproteinsweretested. With a single exceptionproteinLmbC was only involved in interactions which accordingto the number of positive clones (intensivegÍowth.blue color on Leu-glu selectivemedium) appeardisputable(Tab. [). The reasoncould be rhar in the yeast a substantial pafi of the LmbC is in insolubleform as demonstrated Westernblotting, by indicatingthat the heterologous /rnbC expressionis apparently problematic. The LmbC level could be so low that it would be withoutthe detectionlimit or LmbC interactions could be naturallyvery weak. However.almostall the disputable interactionswere found reciprocally. In Tab. 1 interactionsdetectedamong proteins LmbC/LmbE. LmbD/LmbE. LmbF/LmbE, LmblH/LmbE. LmbQ/LmbE and LmbN/LmbE seem ro be mosr important. As LmbE shows homology to a prolein acting on amide bond, its multiple interaction I could suggest that it is a true NDl-svnthetasesubunitand. moreover.a catalyticsubunit directly driving the PPL and MTL condensa(ion. From this point of view, the interaction with LmblH. which has a regulatoryfunction according to Western blot experiments (HOLA et a\.,2003) is also interesting. This interaction could mean thatthereis a direct control(withoutotherintervention) the catalyticalsubunitby a regulatory of protein.The ínteraction LmbE (possiblecatalyticsubunit)with LmbN (showingsimilarity to ACP' of acyl carrier proteins,which in non-ribozomalprotein synthetases serve for transferof growing chains catalytic to centers (ANSARI et a\.,2004) seems be losical. to -9- Lmb protein - AD C D E F IH N o c + + D (+.) + + + E + + + (+) + F + IH (+) + z N o Tab. 1 Results ofpairwise testing ofinteractions among proteins - candidates of NDl-syntetase subunits. - no rnteracnon disputableinteractiont2-3 of 5 clones tested(in one/both * domain combinations)on a selective medium (-Leu-glu) grew well and colonieswereof blue color interactionfound; 3 of 5 clones tested(in oney'both domain í+) combinations) a selectivemedium (.Leu-glu)grew well on and colonieswereof blue color interactionfound:4-5 of 5 clonestested(in one/bothdomain + combinations) a selectivemedium (-Leu-glu)grew well on and colonieswereof blue color In summary,proteinsencodedby the lmbD. lnbE, lmbF and lmbN genescan be consideredvery importantcandidates NDl-synthetasesubunits.LmbD interactswith of LmbC whose relation to the condensationreaction has been already determined experimetallyand which is apparentlya part of the net of interactions formed by the LmbE, LmbF, and LmbN proteíns. Homologsof the proteins(KOBĚRSKA et nl', 2005) can also be found in the gene clustercontrollingbiosynthesis biosynthetically of related celesticetin. can be considered It thatthe proteins could controlcondensation the amino of acid and sugar parts of the antibiotics.Roles of the LmbF and LmbD proteinsis hardly predictable present. at LmbE could play a role of the centralcatalyticsubunit. LmbC role of the PPL activatrngprotein,and LmbN role of ACP. The uhole complex could be controlled LmbQ and LmbIH. by Nevertheless, yeast two-hybridsystem is an artiticlal svstemand the detected the interactionsshouldalso be confirmed othermethods. by l0-
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