The Active Conformation of Avilamycin A Is Conferred by AviX12, a Radical AdoMet Enzyme

Raija Boll,Carsten Hofmann,Björn Heitmann,Gerd Hauser,Steffen Glaser,Thorsten Koslowski,Thorsten Friedrich,Andreas Bechthold

doi:10.1074/jbc.m601508200

Raija Boll, Carsten Hofmann + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m601508200

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Abstract

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.

Highlights

Due to side effects and its poor water solubility, further development was stopped in 2000 [2]
Our results indicated the involvement of AviX12 in the formation of the biologically active conformation of avilamycin A by catalyzing an unusual epimerization reaction
HPLC analysis showed the production of new avilamycin derivatives in comparison to the wild-type strain

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Culture Conditions—DNA manipulation was carried out using E. coli XL-1 Blue MRF (Stratagene) as the host strain. Isolation of E. coli plasmid DNA, DNA restriction, DNA modification, and Southern hybridization were performed following the manufacturer’s directions (Amersham Biosciences, Roche Diagnostics, Promega, and Stratagene). The resulting fragment was isolated with the Nucleospin௡ Extract kit (Macherey Nagel), digested with EcoRI and XbaI, and ligated into pUC18 previously digested with the same enzymes, generating plasmid pUC-aviX12. Construction of pRSET-X12 Expressing AviX12—The aviX12 gene was PCR-amplified using primers 5Ј-AGCCGGAGCTGCAGGCCATATGACCCAG-3Ј (X12ProtF) and 5Ј-CGTCTCGAATTCGTAGGTCTTGCGCAG-3Ј (X12ProtR) containing engineered PstI and EcoRI restriction sites (underlined), respectively. Expression and Purification of AviX12 (N-terminal His6-tagged)—For purification of expressed protein, E. coli strain BL21 (DE3)pLysS cells (Stratagene), carrying either pRSET-X12 with aviX12 or the pRSETb vector alone, were grown in LB broth containing 50 ␮g/ml carbenicillin and 30 ␮g/ml chloramphenicol to an A600 ϭ 0.6. GenBankTM Accession Number—The GenBankTM accession number for the DNA sequence reported in this paper is AAK83189.1

RESULTS

Enterococcus faecium

Hz was found for the

DISCUSSION