Vgb from Staphylococcus aureus inactivates streptogramin B antibiotics by an elimination mechanism not hydrolysis

The streptogramin antibiotics were identified almost 50 years ago but have only recently found clinical use as a consequence of the increase in multid rug-resistant bacteria. Despite the fact that these antibiotics have histor ically not found intense clinical use, resistance to streptogramins exists. Streptogramins consist of a mixture of two components: cyclic polyunsatura ted macrolactones (group A) and cyclic hexadepsipeptides (group B). The lat ter are cyclized through an ester bond between the hydroxyl group of an N-t erminal threonine and the C-terminal carboxyl. Resistance to the B streptog ramins can occur through the production of enzymes such as Vgb from Staphyl ococcus aureus. This enzyme had been assumed to be a lactonase that inactiv ates the cyclic antibiotic by linearization through hydrolytic cleavage of the ester bond. We have expressed recombinant Vgb in quantity and, using a combination of mass spectrometry, NMR, and synthesis of model depsipeptides , show unequivocally that streptogramin B inactivation does not involve hyd rolysis of the ester bond. Rather, the hexadepsipeptide is linearized throu gh an elimination reaction across the ester bond generating an N-terminal d ehydrobutyrine group. Therefore, Vgb is not a hydrolase but a lyase. We als o have explored the activity of Vgb orthologues present in the chromosomes of various bacteria including Bordetella pertussis and Streptomyces coelico lor and have determined that these enzymes also show streptogramin B inacti vation through an elimination mechanism indistinguishable to that used by V gb. These results demonstrate that Vgb is a member of a large group of stre ptogramin B lyases that are present not only in resistant clinical isolates but also in the chromosomes of many bacteria. There is therefore a signifi cant reservoir of streptogramin resistance enzymes in the environment, whic h has the potential to impact the long-term utility of these antibiotics. T his research establishing the molecular mechanism of streptogramin resistan ce therefore has the potential to be exploited in the discovery of inhibito ry compounds that could rescue antibiotic activity even in the presence of resistance elements.