Journal:Acta Cryst F:S1744309112050270

Crystal structure of ADL1, a plant-specific homologue of the universal diaminopimelate amino transferase enzyme of lysine biosynthesis

Vladimir Sobolev, Marvin Edelman, Orly Dym, Tamar Unger, Shira Albeck, Menny Kirma and Gad Galili^[1]

Molecular Tour
The Arabidopsis “Aberrant growth and death-2” (AGD2) protein was originally identified as a protein that is associated with pathogen resistance. The AGD2 protein was subsequently identified as the diaminopimelate amino transferase (DAPAT) enzyme. An Arabidopsis homologue of this protein was subsequently identified and originally named as the AGD2-like defense response protein 1 (ALD1). Both DAPAT and ALD1 require pyridoxal-5′-phosphate (PLP) for their activities. Here we report the structure of the ALD1 protein from Arabidopsis thaliana (AtALD1), to a resolution of 2.3 Å (PDB entry 4fl0). The structure of AtALD1 shows a high level of similarity to the structures described for PLP dependent DAPAT from Arabidopsis thaliana (AtDAPAT) (PDB entry 2z20), with an RMSD between structures of 1.2 Å. AtALD1 is in cyan and AtDAPAT is in yellow. The largest difference between the two structures can be seen at the carboxy terminal regions of two helices: helix α2 (residues 57-73) and helix α12 (residues 393-416). There is a difference of about 6 Å at the loop edge carboxy-terminal to helix α2, between the Cα atom of Asn69 in AtALD1 and the structurally aligned Cα atom of Asp55 in AtDAPAT. The overall binding site structure and position of PLP is similar in AtALD1 and AtDAPAT (RMSD following superimposition equals 0.48 Å over 13 Cα atoms). Carbon atoms of PLP complexed with AtALD1 are in green, carbon atoms of PLP complexed with AtDAPAT are in darkmagenta, nitrogen, oxygen, and phosphorus atoms are colored in blue, red, and orange, respectively. There are, however, two residues that are not conserved: Lys129 and Tyr152 in AtDAPAT versus Gln143 and Phe166 in AtALD1. Lys129 and Tyr152 of AtDAPAT are labeled in yellow and Gln123 and Phe146 in AtALD1 are labeled in cyan. While PLP has contacts with the backbone nitrogen and C-beta atoms of Lys129, it is likely that side chain replacement upon mutation to Gln does not significantly change the position of these atoms and their contacts (click here to see animation of this scene). Furthermore, PLP has only minor contact with the side chain oxygen of Tyr152 (about 1 Ų). Therefore, replacement Tyr by Phe at these positions should not affect PLP binding. Can AtALD1 bind similar substrates as AtDAPAT? For this we: analyzed malate interactions in AtDAPAT (2z1z); compared the structure of the malate binding-site in AtDAPAT with the site formed by the corresponding residues in AtALD1; and speculated on malate interactions if it were placed at the same position in AtALD1 as it has in AtDAPAT. Superposition of the malate binding-site in AtDAPAT with the site formed by the corresponding residues of AtALD1 (RMSD equals 0.71 Å over 10 Cα atoms) revealed that in spite of some rearrangement in the binding site, most contacts are conserved. However, we can see that for the three binding site positions that differ in composition between the structures (Ile63 to Thr77, Lys129 to Gln143 and Tyr152 to Phe166, in AtDAPAT and AtALD1, respectively), malate loses some or all of its contacts in the AtALD1 structure. AtALD1 is in cyan, AtDAPAT is in magenta (2z1z), carbon atoms of PLP complexed with AtALD1 are in green, carbon atoms of PLP complexed with AtDAPAT are in yellow, and carbon atoms of malate are in salmon. In spite of considerable structural similarity between AtALD1 and AtDAPAT, residue differences at the binding site and the resulting changes in putative interaction at the corresponding malate binding positions in AtALD1 lead us to conclude that substrate specificity of AtALD1 is essentially different from those of AtDAPAT. This suggests that either the substrate itself, or the substrate-binding mode, differs in the two proteins, supporting the known in vitro findings.

PDB reference: AtALD1, 4fl0.

References

1. ↑ Sobolev V, Edelman M, Dym O, Unger T, Albeck S, Kirma M, Galili G. Structure of ALD1, a plant-specific homologue of the universal diaminopimelate aminotransferase enzyme of lysine biosynthesis. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Feb 1;69(Pt 2):84-9. doi: , 10.1107/S1744309112050270. Epub 2013 Jan 26. PMID:23385743 doi:10.1107/S1744309112050270