RESUMEN
Glycosyltransferases are key enzymes involved in the biosynthesis of valuable natural products providing an excellent drug-tailoring tool. Herein, we report the identification of two cooperative glycosyltransferases from the sqn gene cluster directing the biosynthesis of saquayamycins in Streptomyces sp. KY40-1: SqnG1 and SqnG2. Gene inactivation of sqnG1 leads to 50-fold decrease in saquayamycin production, while inactivation of sqnG2 leads to complete production loss, suggesting that SqnG2 acts as dual O- and C-glycosyltransferase. Gene inactivation of a third putative glycosyltransferase-encoding gene, sqnG3, does not affect saquayamycin production in a major way, suggesting that SqnG3 has no or a supportive role in glycosylation. The data indicate that SqnG1 and SqnG2 are solely and possibly cooperatively responsible for the sugar diversity observed in saquayamycins 1-7. This is the first evidence of a glycosyltransferase system showing codependence to achieve dual O- and C-glycosyltransferase activity, utilizing NDP-activated d-olivose, l-rhodinose, as well as an unusual amino sugar, presumably 3,6-dideoxy-l-idosamine.
Asunto(s)
Antraquinonas/metabolismo , Carbohidratos/biosíntesis , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Glicosiltransferasas/metabolismo , Streptomyces/enzimología , Conformación de Carbohidratos , Glicosiltransferasas/genéticaRESUMEN
Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.
Asunto(s)
ADN/química , Plicamicina/química , Proteína Proto-Oncogénica c-fli-1/química , Secuencia de Bases , ADN/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Plicamicina/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Proto-Oncogénica c-fli-1/metabolismo , Relación Estructura-ActividadRESUMEN
The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me(2+))-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM=MTM SDK>MTM SA-Trp>MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.