RESUMO
Phaeosphaeride A, a nitrogen-containing bicyclic compound produced by an endophytic fungus, inhibits signaling by the transcription factor STAT3.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Fungos/metabolismo , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Linhagem Celular Tumoral , Compostos Heterocíclicos com 2 Anéis/metabolismo , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacosRESUMO
Meridamycin is a non-immunosuppressive, FKBP12-binding natural macrolide with potential therapeutic applications in a variety of medical conditions. To set the stage for structural modification of meridamycin by genetic engineering, we have cloned and completely sequenced approximately 117 kb of DNA encompassing the meridamycin biosynthetic gene cluster from the producing strain, Streptomyces sp. NRRL 30748. Clustered in the center of the cloned DNA stretch are six genes responsible for the construction of the core structure of meridamycin, including merP encoding a non-ribosomal peptide synthase for pipecolate-incorporation, four PKS genes (merA-D) together encoding 1 loading module and 14 extension modules, and merE encoding a cytochrome P450 monooxygenase. A number of genes with potential pathway-specific regulatory or resistance functions have also been identified. The absence of the gene encoding lysine cyclodeaminase in the sequenced gene cluster and the rest of the genome of NRRL 30748 indicated the synthesis of pipecolate in this strain is not through the common lysine cyclodeamination route previously described for rapamycin and FK506/FK520 biosynthesis. An efficient conjugation method has been developed for Streptomyces sp. NRRL 30748 to facilitate the genetic manipulation of meridamycin biosynthetic gene cluster. Disruption of merP resulted in the complete abolition of meridamycin production, proving the identity of the gene cluster. A novel meridamycin analogue, C36-keto-meridamycin, has been successfully generated through deletion of a DNA fragment encoding KR1 domain of MerA from the chromosomal DNA.
Assuntos
Genes Bacterianos , Macrolídeos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Sondas de DNA/genética , DNA Bacteriano/genética , Macrolídeos/química , Modelos Biológicos , Família Multigênica , Mutagênese InsercionalRESUMO
The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Glicopeptídeos/biossíntese , Glicopeptídeos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Antibacterianos/química , Clonagem Molecular , Cosmídeos , Farmacorresistência Bacteriana , Genes Bacterianos , Glicopeptídeos/química , Glicopeptídeos/genética , Bactérias Gram-Positivas/isolamento & purificação , Modelos Biológicos , Família Multigênica/genética , Oligossacarídeos/biossíntese , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
The mannopeptimycins (MPPs) are potent glycopeptide antibiotics that contain both D and L forms of the unique, arginine-derived amino acid beta-hydroxyenduracididine (betahEnd). The product of the mppO gene in the MPP biosynthetic cluster resembles several non-heme iron, alpha-ketoglutarate-dependent oxygenases, such as VioC and clavaminate synthase. The role of MppO in betahEnd biosynthesis was confirmed through inactivation of mppO, which yielded a strain that produced dideoxy-MPPs, indicating that mppO is essential for generating the beta-hydroxy functionality for both betahEnd residues. Characterization in vitro of recombinant His6-MppO expressed in E. coli revealed that MppO selectively hydroxylates the beta carbon of free L-enduracididine.
