Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Reveal an Unusual Oligomeric Structure.

While humans lack the biosynthetic pathways for -diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from (DapL), a nonpathogenic model bacterium for Complementation experiments verify that the gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an strain that is auxotrophic for -diaminopimelate. Kinetic studies show that DapL follows a Michaelis-Menten mechanism, with a of 4.0 mM toward its substrate l,l-diaminopimelate. The (0.46 s) and the / (115 s M) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that DapL exists in a monomer-dimer self-association, with a of 7.4 μM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in DapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.