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.
Highlights
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
The gene encoding the monofunctional HSD (GmHSD) coding sequence is interrupted by 11 introns that vary in length from 107 to 1637 bp
The obtained results indicate that GmHSD is encoded by a single low copy gene but that hybridization to other HSD-related genes occurs most likely the AK-HSD isoforms in soybean [20]
Summary
The AK-HSD isoforms from Arabidopsis and maize are sensitive to inhibition by threonine [17,18,19, 21], but the enzymes from soybean remain biochemically uncharacterized [20]. To investigate the role of a threonine-resistant HSD in the aspartate amino acid pathway of plants, we have cloned the cDNA and gene encoding the enzyme (GmHSD) from soybean. The isolation of a threonine-resistant HSD from soybean may provide a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content
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