title: Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana.
abstract: 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 micromol of aspartyl phosphate produced min(-1) mg(-1) of protein and 18.8 micromol 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 K(M) values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 microM, 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.
authors.affiliation: Unité Mixte CNRS/INRA/Aventis CropScience, UMR 1932, 14-20 Rue Pierre Baizet, 69263 Lyon, France.
authors.forename: Stephane
authors.initials: S
authors.lastname: Paris
authors.forename: Peter M
authors.initials: PM
authors.lastname: Wessel
authors.forename: Renaud
authors.initials: R
authors.lastname: Dumas
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D001225:Aspartokinase Homoserine Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013912:Threonine; D001225:Aspartokinase Homoserine Dehydrogenase