Structural basis for the evolution of vancomycin resistance D,D-peptidases

Vancomycin resistance in Gram-positive bacteria is due to production of cell-wall precursors ending in d-Ala-d-Lac or d-Ala-d-Ser, to which vancomycin exhibits low binding affinities, and to the elimination of the high-affinity precursors ending in d-Ala-d-Ala. Depletion of the susceptible high-affinity precursors is catalyzed by the zinc-dependent d,d-peptidases VanX and VanY acting on dipeptide (d-Ala-d-Ala) or pentapeptide (UDP-MurNac-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala), respectively. Some of the vancomycin resistance operons encode VanXY d,d-carboxypeptidase, which hydrolyzes both di- and pentapeptide. The molecular basis for the diverse specificity of Van d,d-peptidases remains unknown. We present the crystal structures of VanXY_C and VanXY_G in apo and transition state analog-bound forms and of VanXY_C in complex with the d-Ala-d-Ala substrate and d-Ala product. Structural and biochemical analysis identified the molecular determinants of VanXY dual specificity. VanXY residues 110–115 form a mobile cap over the catalytic site, whose flexibility is involved in the switch between di- and pentapeptide hydrolysis. Structure-based alignment of the Van d,d-peptidases showed that VanY enzymes lack this element, which promotes binding of the penta- rather than that of the dipeptide. The structures also highlight the molecular basis for selection of d-Ala–ending precursors over the modified resistance targets. These results illustrate the remarkable adaptability of the d,d-peptidase fold in response to antibiotic pressure via evolution of specific structural elements that confer hydrolytic activity against vancomycin-susceptible peptidoglycan precursors.The emergence of high-level resistance to vancomycin, a last resort antibiotic against Gram-positive bacteria, in Enterococcus spp. and its spread to methicillin-resistant Staphylococcus aureus is a serious threat to public health (1). Vancomycin acts by binding to the d-alanyl-d-alanine moiety of the uncross-linked N-acetyl-muramyl-l-Ala-d-γ-Glu-l-Lys-d-Ala-d-Ala (pentapeptide[d-Ala]) peptidoglycan precursor blocking the extracellular steps in peptidoglycan synthesis. Resistance is mediated by nine types of operons that replace the d-Ala-d-Ala terminus of peptidoglycan precursors with d-Ala-d-lactate (VanA, -B, -D, and -M types) or d-Ala-d-serine (VanC, -E, -G, -L, and -N types), to which vancomycin exhibits lower binding affinities (2).A critical step in vancomycin resistance involves depletion of d-Ala–terminating precursors to prevent interaction of vancomycin with its target. This step is facilitated by the d,d-dipeptidase VanX and the d,d-pentapeptidase VanY, which hydrolyze, respectively, d-Ala-d-Ala and the C-terminal d-Ala residue from pentapeptide[d-Ala] (3–6). In the d-Ala-d-Ser form of resistance, a single VanXY enzyme evolved to mediate both d,d-dipeptidase and d,d-pentapeptidase activities (7, 8). Thus, Van d,d-peptidases demonstrate variation in peptidoglycan substrate selectivity that correlates with the specific resistance mechanism. As part of the d-Ala-d-Lac type resistance mechanism, the VanX enzyme shows 10⁵-fold higher catalytic efficiency against d-Ala-d-Ala compared with d-Ala-d-Lac substrates, thus facilitating accumulation of the depsipeptide. However, VanX retains significant activity against d-Ala-d-Ser dipeptide (9). Appropriate to its role in resistance, the VanY enzyme shows carboxypeptidase activity against pentapeptide[d-Ala] but lacks activity against d,d-dipeptide substrates (5, 10). The VanXY_C enzyme is selective against resistant dipeptide and pentapeptide peptidoglycan substrates ending in d-Ser (8). Finally, the VanXY_G enzyme, first assigned as a dual substrate active “XY” enzyme by sequence similarity (11), was later shown to lack d,d-pentapeptidase activity typical for bona fide VanXY enzymes (12). The molecular determinants responsible for such diversity of substrate specificity among Van d,d-peptidases are unknown, limiting the understanding of their evolution and hampering the development of inhibitors that could be used in combination with glycopeptides.With the exception of VanY_D, which is a penicillin-binding protein (13), all VanX, VanY, and VanXY d,d-peptidases are zinc-dependent enzymes classified into the metallopeptidase clan MD, family M15, according to the MEROPS database (14). Within clan MD, multiple families of enzymes, including M15 representatives, are involved in bacterial cell wall metabolism (15). Three members of this family have been structurally characterized: zinc d-Ala-d-Ala carboxypeptidase from Streptomyces albus (16) (subfamily M15A), bacteriophage l-Ala-d-Glu peptidase PLY500 (17) (subfamily M15C), and VanX (subfamily M15D) from Enterococcus faecium (18). These enzymes display a common core tertiary fold built on a central antiparallel β-sheet arrayed with multiple α-helices on either face of the β-sheet. The fold contains two consensus motifs that form the active site, His-X(6)-Asp and Glu-X(2)-His, with a zinc ion coordinated by the histidine and aspartate residues. In addition, the structure of VanX revealed a small and constricted active site that explains its specificity toward the d-Ala-d-Ala substrate (18). The VanY and VanXY enzymes belonging to subfamily M15B remain structurally uncharacterized.Given the evolution toward dual substrate specificity of VanXY enzymes, their importance in resistance, and their sequence and functional diversity, we undertook their detailed structural and functional characterization. We determined the crystal structures of VanXY_C and VanXY_G and performed extensive mutagenesis analysis. Our data led to the identification and characterization of the molecular features responsible for their substrate specificities and demonstrated an exceptional diversification and plasticity within their common metallopeptidase fold in response to drug selective pressure.