Figure 1.
The diaminopimelic acid pathway of lysine biosynthesis.
Left: the steps of the pathway are shown with the reactions catalyzed by DapA and DapD indicated by blue and red arrows, respectively. Right: Reactions catalyzed by dihydrodipicolinate synthase (DapA, PA1010) and tetrahydrodipicolinate N-succinyltransferase (DapD, PA3666). The structure of the substrate analogue 2-aminopimelate used as DapD substrate in this study is shown for comparison.
Table 1.
Data collection and refinement statistics of the crystal structures of PaDapD and PaDapA.
Figure 2.
Substrate specificity and reaction kinetics for DapD from P. aeruginosa.
A. Lineweaver – Burk plot for the dependence of DapD on the substrate L-2-aminopimelate. B. Activities of DapD in the presence of L-2-aminopimelate (L-2AP), D-2-aminopimelate (D-2AP) and a racemic mixture of the two compounds (L-2AP+D-2AP) as substrates.
Figure 3.
The structure of P. aeruginosa DapD.
A. Cartoon of the subunit of tetrahydrodipicolinate N-succinyltransferase (DapD). The N-terminal domain is shown in blue, the central domain in green and the C-terminal domain in red. B & C: Views of the trimer of DapD. The three subunits are coloured blue, brown and yellow. D. Surface illustration of the trimer of the DapD-coenzyme A-succinate complex. The substrate binding grooves are formed between the left handed β-helix domains from adjacent subunits (blue and brown, respectively) of the trimer. Bound co-enzyme A and succinate are shown as stick models in yellow. E. Interactions of DapD with the bound coenzyme-A and succinate. The two subunits contributing to this active site are shown in blue and brown, respectively. Coenzyme-A is shown in yellow sticks and succinate in orange, while the amino acid side chains involved in the interaction are depicted in light gray.
Figure 4.
Evolutionary tree of DapD enzymes.
The tree is based on a sequence alignment generated by the program Phylogenetic Tree available at http:www.cbrg.ethz.ch/services. Known crystal structures are added as illustrations to protein fold-evolution. The N-terminal domains are shown in blue, the central LβH-domains in orange and the C-terminal domains in red.
Figure 5.
A & B: OMIT electron density maps [44] for bound ligands L-2AP (A) and D-2AP (B), contoured at 1.6σ. C & D: Interactions of L-2AP (C) and D-2AP (D) at the active site of PaDapD. The two adjacent subunits that form an active site cleft are shown in blue and brown colours. L-2AP is depicted in blue and D-2AP in green. Enzyme residues interacting with the ligands are shown in light grey.
Figure 6.
Assessment of the effect of PA1010 gene deletion in vivo.
Quantification of P. aeruginosa colonies grown in the lung of mice intratracheally infected with agarose beads loaded with the P. aeruginosa gene knock-out mutant (ΔPA1010) in comparison to PAO1 wildtype (wt).
Figure 7.
The structure of P. aeruginosa DapA.
A. Size exclusion chromatography elution profile of PaDapA (PA1010) indicating that a single species exists in solution. Based on the calibration curve (insert) the calculated molecular mass is 60 kDa. B. PaDapA (PA1010) purified sample analyzed in native polyacrylamide gel electrophoresis indicating a single species. C. Stereo view of the active site of PaDapA located in the center of the α/β barrel. Amino acid side chains forming the active site are indicated as stick models. Residues conserved in the three homologues PA1010, PA0223 and PA4188 are shown in yellow, while the variable positions Thr44, Arg138 and Lys109 are indicated in purple. D. Sequence conservation in DapA enzymes from Escherichia coli, Bacillus anthracis, Pseudomonas aeruginosa and the proposed DapA paralogues in the PAO1 genome PA0223 and PA4188. The active site residues in PaDapA (PA1010) are indicated with yellow or purple colour as in C.
Figure 8.
Structural basis of inhibition by D-2-aminopimelate.
A. Composite stereo view of the active site of PaDapD with bound succinamide-CoA, and the substrate L-2-AP. The position of the inactive substrate analogue succinamide-CoA (orange) and the L-2-aminopimelate (yellow) bound in the ternary complex were derived from a superposition with the DapD-succinamide-CoA-L-2AP complex (1KGQ). The bound L-2AP in the PaDapD-L-2AP complex is shown in blue. B. View of a composite model of the catalytically incompetent complex of PaDapD with bound succinamide-CoA, and the inhibitor D-2AP. The model was created using the same templates as in (A), the bound D-2AP in the structure of the complex of PaDaD with this ligand is shown in green.