Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase.

The streptogramin combination therapy of quinupristin-dalfopristin (Synercid) is used to treat infections caused by bacterial pathogens, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. However, the effectiveness of this therapy is being compromised because of an increased incidence of streptogramin resistance. One of the clinically observed mechanisms of resistance is enzymatic inactivation of the type B streptogramins, such as quinupristin, by a streptogramin B lyase, i.e., virginiamycin B lyase (Vgb). The enzyme catalyzes the linearization of the cyclic antibiotic via a cleavage that requires a divalent metal ion. Here, we present crystal structures of Vgb from S. aureus in its apoenzyme form and in complex with quinupristin and Mg2+ at 1.65- and 2.8-A resolution, respectively. The fold of the enzyme is that of a seven-bladed beta-propeller, although the sequence reveals no similarity to other known members of this structural family. Quinupristin binds to a large depression on the surface of the enzyme, where it predominantly forms van der Waals interactions. Validated by site-directed mutagenesis studies, a reaction mechanism is proposed in which the initial abstraction of a proton is facilitated by a Mg2+ -linked conjugated system. Analysis of the Vgb-quinupristin structure and comparison with the complex between quinupristin and its natural target, the 50S ribosomal subunit, reveals features that can be exploited for developing streptogramins that are impervious to Vgb-mediated resistance.

Mycobacterium smegmatis Erm(38) is a reluctant dimethyltransferase.

The waxy cell walls of mycobacteria provide intrinsic tolerance to a broad range of antibiotics, and this effect is augmented by specific resistance determinants. The inducible determinant erm(38) in the nontuberculous species Mycobacterium smegmatis confers high resistance to lincosamides and some macrolides, without increasing resistance to streptogramin B antibiotics. This is an uncharacteristic resistance pattern falling between the type I and type II macrolide, lincosamide, and streptogramin B (MLS(B)) phenotypes that are conferred, respectively, by Erm monomethyltransferases and dimethyltransferases. Erm dimethyltransferases are typically found in pathogenic bacteria and confer resistance to all MLS(B) drugs by addition of two methyl groups to nucleotide A2058 in 23S rRNA. We show here by mass spectrometry analysis of the mycobacterial rRNA that Erm(38) is indeed an A2058-specific dimethyltransferase. The activity of Erm(38) is lethargic, however, and only a meager proportion of the rRNA molecules become dimethylated in M. smegmatis, while most of the rRNAs are either monomethylated or remain unmethylated. The methylation pattern produced by Erm(38) clarifies the phenotype of M. smegmatis, as it is adequate to confer resistance to lincosamides and 14-member ring macrolides such as erythromycin, but it is insufficient to raise the level of resistance to streptogramin B drugs above the already high intrinsic tolerance displayed by this species.

Chemoenzymatic approach to enantiopure streptogramin B variants: characterization of stereoselective pristinamycin I cyclase from Streptomyces pristinaespiralis.

Streptogramin B antibiotics are cyclic peptide natural products produced by Streptomyces species. In combination with the synergistic group A component, they are "last line of defense" antimicrobial agents against multiresistant cocci. The racemization sensitivity of the phenylglycine (Phg(7)) ester is a complex challenge in total chemical synthesis of streptogramin B molecules. To provide fast and easy access to novel streptogramin antibiotics, we introduce a novel chemoenzymatic strategy in which diversity is generated by standard solid phase protocols and stereoselectivity by subsequent enzymatic cyclization. For this approach, we cloned, overproduced, and biochemically characterized the recombinant thioesterase domain SnbDE TE of the pristinamycin I nonribosomal peptide synthetase from Streptomyces pristinaespiralis. SnbDE TE catalyzes regioselective ring closure of linear peptide thioester analogues of pristinamycin I as well as stereoselective cyclization out of complex in situ racemizing substrate mixtures, enabling synthesis of Streptogramin B variants via a dynamic kinetic resolution assay. A remarkable substrate tolerance was detected for the enzymatic cyclization including all the seven positions of the peptide backbone. Interestingly, SnbDE TE was observed to be the first cyclase from a macrolactone forming NRPS which is additionally able to catalyze macrolactamization of peptide thioester substrates. An N-methylated peptide bond between positions 4 and 5 is mandatory for a high substrate turnover. The presented strategy is potent to screen for analogues with improved activity and guides our understanding of structure--activity relationships in the important class of streptogramin antibiotics.