Overproduction, Purification, and Characterization of Recombinant Bifunctional Threonine-Sensitive Aspartate Kinase-Homoserine Dehydrogenase from Arabidopsis thaliana

In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis thaliana). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 μmol of aspartyl phosphate produced min−1 mg−1 of protein and 18.8 μmol of NADPH formed min−1 mg−1 of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with KM values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 μM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa.