Vancomycin Derivative with Damaged d-Ala-d-Ala Binding Cleft Binds to Cross-linked Peptidoglycan in the Cell Wall of Staphylococcus aureus

Biochemistry, 2008, 47 (12), pp 3822--3831

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-13C]alanine, [1-13C]glycine, and l-[e-15N]lysine. Internuclear distances from 19F of the DFPBV to the 13C and 15N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The 13C{19F} and 15N{19F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).

Chemical structures of some second-generation glycopeptide antibiotics (right) and their parent compounds (left). Synthetic modifications of the second-generation glycopeptides are highlighted.

Chemical structures of vancomycin and N-4-(4-fluorophenyl)benzyl-vancomycin (left) and their corresponding Edman degradation products (right). Terminal leucyl residues are highlighted.

13C{19F}REDOR dephasing (DS/S0) as a function of the dipolar evolution time for a complex of DFPBV with whole cells of S. aureus labeled by d-[1-13C]alanine. The dephasing was measured for two values of the static magnetic field. Representative error bars based on integrals (see Figure 4) were determined by the uncertainties in DS, which range from +-15% near 5 ms evolution to+-5% at 20 ms evolution. The total number of scans accumulated was 2,018,656 for both S and S0 spectra. This accumulation required 93 days of spectrometer time. The solid line shows the calculated dephasing for a single 13C-19F distance of 7.2 A and a maximum dephasing of 1.6%.

Two-dimensional schematic representation of peptidoglycan assembly in S. aureus. Chain extension (from right to left) at the active site of transglycosylase (white oval) and cross-linking at the active site(s) of transpeptidase (white circles) are synchronized. This ensures that the orientation of monomers added to the growing nascent peptidoglycan is determined by the template strand (the last completed glycan chain shown in light gray) to which they ultimately will be cross-linked. Glycopeptides, such as DFPBV (pink-purple-green), cannot bind to d-Ala-d-Ala and therefore are unlikely to bind to lipid II (yellow) but can bind to mature peptidoglycan or between mature and template strands (bottom, left) at sites that are not cross-linked and thus are sterically free. The 19F (green) of the hydrophobic substituent (blue) at such sites is near the d-alanine (red) of a neighboring stem. DFPBV can also bind at structurally similar template sites, which are adjacent to nascent peptidoglycan, and this leads to inhibition of cross-linking. DFPBV possibly binds at nascent peptidoglycan sites, which could lead to the inhibition of chain extension.