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Dihydropyrrole Formation During Sibiromycin Biosynthesis

dc.contributor.advisorRokita, Steven E.en_US
dc.contributor.authorSaha, Shalinien_US
dc.date.accessioned2015-09-18T06:03:10Z
dc.date.available2015-09-18T06:03:10Z
dc.date.issued2015en_US
dc.identifierhttps://doi.org/10.13016/M2QM0C
dc.identifier.urihttp://hdl.handle.net/1903/17112
dc.description.abstractA description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis in actinomycetes is a prerequisite for engineering production of analogs with enhanced antitumor activity. Several proteins expected to synthesize the PBD’s dihydropyrrole moiety were heterologously expressed, purified and assayed for activity. UV-visible spectroscopy revealed that predicted dioxygenases SibV and homolog Orf12 associated with PBDs sibiromycin and anthramycin, respectively, catalyze the regiospecific 2,3-extradiol dioxygenation of L-3,4-dihydroxyphenylalanine (L-DOPA) to form L-2,3-secodopa (λmax = 368 nm). 1H NMR spectroscopy indicated that L-2,3-secodopa then spontaneously cyclizes into the α-keto acid tautomer of 4-(2-oxo-3-butenoic-acid)-4,5-dehydro-L-proline 1.1 (λmax = 414 nm). Thus, the dioxygenases establish the scaffold of the dihydropyrrole moiety. Both the quaternary structure and product formed by dioxygenases are conserved in dihydropyrrole biosynthesis within both PBD and non-PBD pathways. Stability studies suggest that 1.1 is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. Hydrolysis and methylation steps were proposed to modify the dihydropyrrole scaffold in PBDs. The predicted proteins SibS and homolog TomK associated with sibiromycin and PBD tomaymycin biosynthesis, respectively, were assayed for hydrolysis activity. The predicted protein SibZ associated with sibiromycin biosynthesis was assayed for methyltransferase activity. The proposed SibZ substrate 4-vinyl-4,5-dehydro-L-proline 1.2 was synthesized. For these three proteins, no catalytic activity was observed with their proposed substrates or substrate precursors under a range of conditions. However, SibS binds 1.1 (KD = 64 ± 2 µM) suggesting it participates in dihydropyrrole biosynthesis. HPLC-MS indicated that SibS catalyzes the depurination of S-adenosylmethionine although it is unlikely this reaction is involved in sibiromycin biosynthesis. These findings suggest that the pathway requires revision. The adenylation and thiolation didomain of the predicted non-ribosomal peptide synthetase SibD associated with sibiromycin biosynthesis was expressed and purified. To test if SibD incorporates the dihydropyrrole moiety into sibiromycin, its proposed substrate 4-propenyl-4,5-dehydro-L-proline 1.5 was synthesized. A radioactivity exchange assay and peptide analysis by MS revealed that SibD does not adenylate and thiolate 1.5 or its precursors L-DOPA, 1.1 or 1.2. However, L-threonine and the metabolic precursor L-tyrosine are substrates for these reactions. SibB promotes adenylation catalyzed by SibD and represents one of two proteins, distinct from MbtH-like proteins, capable of promoting adenylation.en_US
dc.language.isoenen_US
dc.titleDihydropyrrole Formation During Sibiromycin Biosynthesisen_US
dc.typeDissertationen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.contributor.departmentChemistryen_US
dc.subject.pqcontrolledChemistryen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledBiosynthesisen_US
dc.subject.pquncontrolledDihydropyrroleen_US
dc.subject.pquncontrolledEnzymeen_US
dc.subject.pquncontrolledNatural productsen_US
dc.subject.pquncontrolledNonribosomal peptide synthetaseen_US
dc.subject.pquncontrolledPyrrolobenzodiazepinesen_US


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