Figure 1.
DAP/L-lysine anabolic pathways.
The pathways are denoted by the acyl pathways (1), l,l-diaminopimelate aminotransferase pathway (2) and the meso-DAP dehydrogenase pathway (3). The abbreviations of the enzymes are as follows: tetrahydrodipicolinate acylase (DapD), acyl-amino-ketopimelate aminotransferase (DapC), acyl-ketopimelate deacylase (DapE), diaminopimelate epimerase (DapF), diaminopimelate decarboxylase (LysA), m-diaminopimelate dehydrogenase (Ddh) and l,l-diaminopimelate aminotransferase (DapL).
Figure 2.
Recombinant expression and purification of Cr-DapL from E. coli.
Lane (1)–Protein marker (kDa), Lane (2)–10 µg uninduced soluble protein extract, Lane (3)–10 µg induced soluble extract, Lane (4)–10 µg of post binding protein, Lane (5)–1.0 µg pure recombinant Cr-DapL protein. The proteins were resolved on a SDS-PAGE gel containing 10% (w/v) acrylamide and the gel was stained using Coomassie Blue.
Table 1.
Substrate specificity of Cr-DapL using four amino donors.
Table 2.
Kinetic properties of Cr-DapL.
Table 3.
Hydrodynamic properties of Cr-DapL.
Figure 3.
Functional complementation of the E. coli dapD/E mutant.
Functional complementation was tested using the E. coli dapD/E double mutant (AOH1). The plasmids pBAD33 and pBAD33+Cr-DapL were selected on LB agar medium supplemented with 50 µg mL−1 DAP and 34 µg mL−1 chloramphenicol. The bacteria were grown to an OD of 0.5 at 600 nm, the strain were serially diluted to 10−1, 10−2, 10−3 and 10−4 using 0.85% (w/v). The strain harboring the pBAD33 and pBAD33+Cr-DapL was replica-plated onto LB medium plus 0.2% (w/v) arabinose with or without 50 µg mL−1 DAP. The cultures were grown at 30 °C for 24 hours.
Figure 4.
The wavelength scans were performed between 240 and 195 nm. The scan was performed at a Cr-DapL concentration of 1 µM. The final spectrum (□) is the average result from three scans taken at 20°C. The CONTIN algorithm from the CDpro software package produced the best fit (solid line) against the SP43 protein database [25] with an r.m.s.d. = 0.18 M−1 cm−1. The fit predicts ∼50% α-helix content, ∼15% β-sheet, ∼15% turn, and ∼20% unordered.
Table 4.
Refinement statistics for the crystal structure of Cr-DapL.
Figure 5.
Sedimentation velocity analysis of Cr-DapL at 9.2 µM.
A) Continuous mass, c(M), distribution is plotted for Cr-DapL (solid line), suggesting a mass of ∼100 kDa. The predicted mass of the dimer is 97.66 kDa. Analysis was performed using the program SEDFIT [26], [27] at a resolution of 200, with massmin = 10 kDa, massmax = 180 kDa and at a confidence level (F-ratio) = 0.95. Statistics for the nonlinear least squares best fits were r.m.s.d. = 0.005, runs test-Z = 3. Residuals (B) for the c(M) distribution best fits (C) plotted as a function of radial position (cm) from the axis of rotation for Cr-DapL at 9.2 µM.
Figure 6.
The crystal structure of Cr-DapL.
A) The dimer in the asymmetric unit. This view looks down the non-crystallographic two-fold axis. B) An overlay of the Cr-DapL dimer (magenta) with that of the apo-Ar-DapL (3EI7, yellow). The r.m.s.d. for the overlay was 0.67 Å for the α-carbon atoms. C) Monomer structure with the domains highlighted in the stereo image (D).
Figure 7.
Location and orientation of the active-site of Cr-DapL.
A) Location of the two active-sites in the dimer, as highlighted by the position of a PLP molecule, taken from an overlay of Cr-DapL with Ar-DapL+PLP structure (2Z20),shown in stick form (magenta). PLP was not found in the active site of Cr-DapL. B) Stereoview of the active-site showing the loops that contribute residues to the active-site. Again PLP is added to the structure from an overlay with Ar-DapL+PLP structure (2Z20). The image overlays the monomers of Cr-DapL (blue and red) with that of the apo-Ar-DapL (yellow and green). In B) and C), the asterisk emphasizes loops that are contributed from the opposing monomer in the dimer. C) Bonding of residues in loops A and C* with the sulfate, which sits in the same position as the phosphate of PLP.
Figure 8.
Overlay active site residues of Cr-DapL with apo-Ar-DapL.
Stereoview of the putative active-site residues conserved between Cr-DapL and Ar-DapL (see sequence and structural alignments in Supplementary Figure 3). As in Figure 7, the asterisk emphasizes residues that are contributed from the opposing monomer in the dimer. Numbering is based on the Cr-DapL structure. The sulfate and PLP molecules are also shown.
Figure 9.
Analysis of T-DNA mutant line SAIL_208_H11.
A) PCR analysis of the SAIL_208_H11 Arabidopsis T-DNA mutant line: Lane (1)–DNA ladder (base pairs), Lane (2)-negative control, Lane (3)-WT-non transgenic plant, Lane (4)-WT-segregant, Lane (5)-heterozygous plant. B) Schematic localization of the T-DNA insertion site, which is located in the 5′ UTR of the gene. C) Phenotype analysis of a heterozygous silique showing the WT seed (black arrow) and mutant or aborted seeds (white arrows).