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 multidrug-resistant bacteria. Despite the fact that these antibiotics have historically not found intense clinical use, resistance to streptogramins exists. Streptogramins consist of a mixture of two components: cyclic polyunsaturated macrolactones (group A) and cyclic hexadepsipeptides (group B). The latter are cyclized through an ester bond between the hydroxyl group of an N-terminal threonine and the C-terminal carboxyl. Resistance to the B streptogramins can occur through the production of enzymes such as Vgb from Staphylococcus aureus. This enzyme had been assumed to be a lactonase that inactivates 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 hydrolysis of the ester bond. Rather, the hexadepsipeptide is linearized through an elimination reaction across the ester bond generating an N-terminal dehydrobutyrine group. Therefore, Vgb is not a hydrolase but a lyase. We also have explored the activity of Vgb orthologues present in the chromosomes of various bacteria including Bordetella pertussis and Streptomyces coelicolor and have determined that these enzymes also show streptogramin B inactivation through an elimination mechanism indistinguishable to that used by Vgb. These results demonstrate that Vgb is a member of a large group of streptogramin B lyases that are present not only in resistant clinical isolates but also in the chromosomes of many bacteria. There is therefore a significant reservoir of streptogramin resistance enzymes in the environment, which has the potential to impact the long-term utility of these antibiotics. This research establishing the molecular mechanism of streptogramin resistance therefore has the potential to be exploited in the discovery of inhibitory compounds that could rescue antibiotic activity even in the presence of resistance elements.