Threonine-insensitive Homoserine Dehydrogenase from Soybean

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 ϳ150 M). 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.

Structural basis for the catalytic mechanism of homoserine dehydrogenase

“…The enzymatic activity of S. aureus HSD was evaluated using previously described protocols (Schroeder et al, 2010). Briefly, 100 ml of the reaction mixture contained 30 mM l-homoserine (Sigma-Aldrich) and varying concentrations of the co-substrate NADP + (Hi-Media Inc.).…”

“…The sequence features and lengths of the -strands in HSD contribute to structural differences from the lactate dehydrogenase (LDH)-like Rossmann fold. The catalytic domain is comprised of an antiparallel -sheet that is involved in dimerization and an -helical cluster that contains the active site (DeLaBarre et al, 2000;Schroeder et al, 2010).…”

“…Monofunctional HSDs are homodimers and are mostly found in fungi, archaea, Gram-positive bacteria and certain Gram-negative bacteria. Bifunctional HSDs (fused to aspartokinase at the N-terminus), on the other hand, adopt a tetrameric arrangement (Archer et al, 1991;Parsot & Cohen, 1988;Schroeder et al, 2010;Thomas et al, 1993). In a ISSN 1399-0047 # 2015 International Union of Crystallography few variants of monofunctional HSDs, primarily from Grampositive and certain Gram-negative bacteria, the catalytic domain is fused to an additional regulatory domain (ACT domain) at the C-terminus (Curien et al, 2008).…”

“…HSD follows an ordered bi-bi kinetic mechanism, in which NADPH binding precedes the binding of ASA at the active site, whereas the product, l-Hse, is released first followed by NADP + (Jacques, Ejim et al, 2001;Schroeder et al, 2010). Two probable reaction mechanisms have been proposed for the forward reaction of HSD (DeLaBarre et al, 2000).…”

Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases

“…Subsequent metabolism of Hse yields methionine, threonine or isoleucine, which are all essential in humans. Because HseDH is central to the synthesis of these amino acids, regulation of the enzyme by its end products has been extensively studied in bacteria, yeast and plants at both the activity and genetic levels 4 5 6 7 . In particular, threonine-dependent feedback regulation to control HseDH activity has been analyzed in detail.…”