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

Abstract

VanY(n) 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. VanY(n) (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY(n) 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 Zn(2+) and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY(n) as in VanY, VanX and VanXY Zn(2+)-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, VanY(n) shows D,D-carboxypeptidase and D,D-dipeptidase activity, but lacks D,D-carboxyesterase ability on D-Ala-D-Lac-terminating peptides. VanY(n) belongs to the metallo-D,D-carboxypeptidase family, but it is inhibited by β-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.