Characterization of VanYn, a novel d,d-peptidase/d,d-carboxypeptidase involved in glycopeptide antibiotic resistance in Nonomuraea sp ATCC 39727

摘要

VanYn is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase III of clinical development. VanYn (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His 6-tagged VanYn was successfully expressed as a soluble and active protein in Escherichia coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn2+ and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanYn as in VanY, VanX and VanXY Zn2+-dependent d,d-carboxypeptidases and d-Ala-d-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanYn shows d,d-carboxypeptidase and d,d-dipeptidase activity, but lacks d,d-carboxyesterase ability on d-Ala-d-Lac-terminating peptides. VanYn belongs to the metallo-d,d-carboxypeptidase family, but it is inhibited by beta-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation glycopeptide such as dalbavancin. Database The nucleotide sequence of vanYn is available in the GenBank data base under accession number CAD91202