Assuntos
Genes Bacterianos/genética , Streptomyces/genética , Streptomyces/metabolismo , Ativação Transcricional/genética , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/efeitos dos fármacos , Mutação/fisiologia , Oligossacarídeos/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The antibiotic avilamycin A is produced by Streptomyces viridochromogenes Tü57. Avilamycin belongs to the family of orthosomycins with a linear heptasaccharide chain linked to a terminal dichloroisoeverninic acid as aglycone. The gene cluster for avilamycin biosynthesis contains 54 open reading frames. Inactivation of one of these genes, namely aviX12, led to the formation of a novel avilamycin derivative named gavibamycin N1. The structure of the new metabolite was confirmed by mass spectrometry (MS) and NMR analysis. It harbors glucose as a component of the heptasaccharide chain instead of a mannose moiety in avilamycin A. Antibacterial activity tests against a spectrum of Gram-positive organisms showed that the new derivative possesses drastically decreased biological activity in comparison to avilamycin A. Thus, AviX12 seems to be implicated in converting avilamycin to its bioactive conformation by catalyzing an unusual epimerization reaction. Sequence comparisons grouped AviX12 in the radical S-adenosylmethionine protein family. AviX12 engineered with a His tag was overexpressed in Escherichia coli and purified by affinity chromatography. The iron sulfur cluster [Fe-S] present in radical AdoMet enzymes was detected in purified AviX12 by means of electron paramagnetic resonance spectroscopy.
Assuntos
Racemases e Epimerases/química , S-Adenosilmetionina/química , Configuração de Carboidratos , Espectroscopia de Ressonância de Spin Eletrônica , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Dados de Sequência Molecular , Oligossacarídeos/química , Fases de Leitura Aberta , Polissacarídeos/química , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Conformação ProteicaRESUMO
The oligosaccharide antibiotic avilamycin A is composed of a polyketide-derived dichloroisoeverninic acid moiety attached to a heptasaccharide chain consisting of six hexoses and one unusual pentose moiety. We describe the generation of mutant strains of the avilamycin producer defective in different sugar biosynthetic genes. Inactivation of two genes (aviD and aviE2) resulted in the breakdown of the avilamycin biosynthesis. In contrast, avilamycin production was not influenced in an aviP mutant. Inactivation of aviGT4 resulted in a mutant that accumulated a novel avilamycin derivative lacking the terminal eurekanate residue. Finally, AviE2 was expressed in Escherichia coli and the gene product was characterized biochemically. AviE2 was shown to convert UDP-D-glucuronic acid to UDP-D-xylose, indicating that the pentose residue of avilamycin A is derived from D-glucose and not from D-ribose. Here we report a UDP-D-glucuronic acid decarboxylase in actinomycetes.
Assuntos
Carboxiliases/biossíntese , Carboxiliases/genética , Escherichia coli/enzimologia , Oligossacarídeos/biossíntese , Pentoses/biossíntese , Actinobacteria/enzimologia , Sequência de Carboidratos , Escherichia coli/genética , Glucose/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/análise , Oligossacarídeos/metabolismoRESUMO
The oligosaccharide antibiotics avilamycin A and C are produced by Streptomyces viridochromogenes Tu57. Both consist of a heptasaccharide chain, which is attached to a polyketide-derived dichloroisoeverninic acid moiety. They show excellent antibiotic activity against Gram-positive bacteria. Both molecules are modified by O-methylation at different positions, which contributes to poor water solubility and difficulties in galenical drug development. In order to generate novel avilamycin derivatives with improved polarity and improved pharmacokinetic properties, we generated a series of mutants with one, two, or three mutated methyltransferase genes. Based on the structure of the novel avilamycin derivatives, the exact function of three methyltransferases, AviG2, AviG5, and AviG6, involved in avilamycin biosynthesis could be assigned.