Crystal structure and mechanism of the Staphylococcus cohnii virginiamycin B lyase (Vgb).
The semisynthetic streptogramin antibiotic quinupristin/dalfopristin (trade name Synercid, Aventis Pharma) is a mixture of the A-type streptogramin dalfopristin and the B-type streptogramin quinupristin, a capped hexapeptide macrolactone. Quinupristin/dalfopristin was developed to combat multidrug resistant pathogens, but suffers from its own problems with drug resistance. Virginiamycin B lyase (Vgb) inactivates the quinupristin component of Synercid by lactone ring opening. Remarkably, the enzyme promotes this reaction by intramolecular beta-elimination without the involvement of a water molecule. Recently, structures of S. aureus Vgb in the presence and absence of substrate were reported and used together with detailed mutagenesis data to suggest a catalytic mechanism. Here, we report an independent determination of the S. cohnii Vgb crystal structure and a biochemical characterization of the enzyme. As expected, the S. cohnii and S. aureus Vgb structures and active sites are very similar. Moreover, both enzymes catalyze quinupristin lactone ring opening with similar rate constants, albeit perhaps with different dependencies on divalent metal ions. Replacement of the conserved active site residues His228, Glu268, or His270 with alanine reduces or abolishes S. cohnii Vgb activity. Residue Lys285 in S. cohnii Vgb is spatially equivalent to the S. aureus Vgb active site residue Glu284. A glutamate but not an alanine residue can substitute for the lysine without significant loss of activity.
Lipka
Magdalena
M
International Institute of Molecular and Cell Biology, Trojdena Street 4, 02-109 Warsaw, Poland.
Filipek
Renata
R
Bochtler
Matthias
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D006420:Hemeproteins; C100771:Vgb protein, Bacteria