Figure 1.
Overall structure of NDM-1 and its sequence alignment with its homologue proteins.
A. Sequence alignment of NDM-1 with VIM-4, FEZ-1 and CphA. The second structure assignment of VIM-4 is labeled on the top of the sequences. Black quadrangles indicate residues that coordinate with Zn2+(I), while black circles indicate residues that coordinate with Zn2+(II). The residues composing of the loop1 of NDM-1 is labeled in green box. B. Cartoon representation of the overall structure of NDM-1 is in light orange color. The loop1, loop2 and the insertion are colored in red, the conserve residues coordinating with zinc ions are represented as sticks in gray color, and the zinc ions shown as spheres.
Figure 2.
Molecular models of NDM-1 and its complex with antibiotics.
A. The active site of NDM-1 with two zinc ions and the coordinating residues. B. The comparison of the two loops in NDM-1 and VIM-2. The loops and key residues in NDM-1 are colored in gray, while in VIM-2 colored in green. C. The binding modes of antibiotics imipenem and carbapenem in the active site of NDM-1. The two antibiotics are colored in orange and yellow sticks respectively, while the key residues in gray sticks. The lactam motifs are all colored in green.
Figure 3.
One proposed bis-zinc-form mechanism for the hydrolysis of cephalosporin scaffold by B1 subclass enzyme NDM-1.
Figure 4.
Complex models comparison between NDM-1(A), VIM-2(B) and FEZ-1(C).
Upper panel shows the structural electrostatic surfaces (the color blue indicates the positive-charge, while red negative-charged), two zinc ions are shown as gray spheres, and antibiotics meropenem is represented in green sticks. The detailed interactions between meropenem and three enzymes NDM-1 (gray sticks), VIM-2 (yellow sticks) and FEZ-1 (orange sticks) are shown on the lower panel.
Figure 5.
Binding mode of hydrolyzed meropenem with NDM-1.
The left displays the structural electrostatic surface and the right is the detailed binding interactions.
Figure 6.
NDM-1 protein expression and purification.
A. 6×His and sumo (small ubiquitin-related modifier) tagged NDM-1 was overexpressed in E.coli BL21(DE3) strain, the black arrow indicates the sumo-NDM-1 fusion protein. B. The cleaved NDM-1 protein was purified by S75 gel filtration chromatography. C. SDS-PAGE gel shows the purity of NDM-1 more than 95%.
Figure 7.
The chemical structures of the seven antibiotics tested in the assay of NDM-1 catalytic activity.
Figure 8.
Hydrolysis activity of NDM-1 by enzymatic assays.
A. Seven antibiotics were hydrolyzed by NDM-1 protein. The enzymatic reactions were dynamically monitored by the degradation of the antibiotics. C0 is the initial concentration of antibiotics. Error bars, s.d. B. Tigecycline inhibited the hydrolysis activity of NDM-1 to meropenem weakly. Compare with control (black line), 50 µM (red line) and 150 µM (blue line) tigecycline slowed down the hydrolysis velocity of meropenem by NDM-1. C0 is the initial concentration of meropenem. Error bars, s.d. C. Compare with the wild type enzyme (pink line), the point mutants C208A (black line), D124A (red line), K211A (green line) and K211E (blue line) totally disrupt the hydrolytic activity to meropenem; while mutations of loop1 (dark yellow line) hardly affect the NDM-1 hydrolytic activity. C0 is the initial concentration of meropenem. Error bars, s.d.