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Abstract

In some bacterial species and photosynthetic cohorts, including algae, the enzyme l,l-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to l,l-diaminopimelate (l,l-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and l,l-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid l-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

Kinetic properties of Cr-DapL

The pure recombinant enzyme was used to perform enzyme assays to assess the kinetic properties using forward and reverse quantitative assays. In the reverse assay, L,L-DAP serves as the amino acceptor and 2-ketoglutarate serve as the amino acceptor. In the anabolic direction of l-lysine synthesis, glutamate serves as the amino donor and THDPA serves as the amino acceptor. Using these assays, the kinetic properties of the enzyme were tested at varying concentrations of one substrate and at saturation levels of other substrates (Supplementary ). The reciprocal plots were linear and were consistent with Michaelis-Menten kinetics. The V max for the forward and reverse directions were calculated along with the apparent K M for the various substrates. The enzyme has a maximum velocity of approximately 11.6 µmol min−1 mg−1 in the reverse direction and 0.68 µmol min−1 mg−1 in the forward direction (). The apparent K M for the four substrates were 0.3 mM for L,L-DAP, 2.2 mM for 2-ketoglutarate, 0.10 mM for THDPA and 0.9 mM for glutamate.. The kinetic properties of Cr-DapL are comparable to the Arabidopsis ortholog that was previously characterized ().