title: Threonine-insensitive homoserine dehydrogenase from soybean: genomic organization, kinetic mechanism, and in vivo activity.
abstract: Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K(i) = 160-240 mM) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K(i) approximately 150 microM). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.
authors.affiliation: Department of Biology, Washington University, St. Louis, Missouri 63130, USA.
authors.forename: Amy C
authors.initials: AC
authors.lastname: Schroeder
authors.forename: Chuanmei
authors.initials: C
authors.lastname: Zhu
authors.forename: Srinivasa Rao
authors.initials: SR
authors.lastname: Yanamadala
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Cahoon
authors.forename: Kiani A J
authors.initials: KA
authors.lastname: Arkus
authors.forename: Leia
authors.initials: L
authors.lastname: Wachsstock
authors.forename: Jeremy
authors.initials: J
authors.lastname: Bleeke
authors.forename: Hari B
authors.initials: HB
authors.lastname: Krishnan
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D006715:Homoserine Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics*; D013912:Threonine / Q000737:chemistry
chemical_list: D010940:Plant Proteins; D013912:Threonine; D006715:Homoserine Dehydrogenase