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Title: Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity

item SCHROEDER, AMY - Washington University
item ZHU, CHUANMEI - Washington University
item YANAMADALA, SRINIVASA RAO - University Of Missouri
item CAHOON, REBECCA - Danforth Plant Science Center
item ARKUS, KIANI - Washington University
item WACHSSTOCK, LEIA - Danforth Plant Science Center
item BLEEKE, JEREMY - Danforth Plant Science Center
item Krishnan, Hari
item JEZ, JOSEPH - Washington University

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2009
Publication Date: 11/6/2009
Citation: Schroeder, A.C., Zhu, C., Yanamadala, S., Cahoon, R.E., Arkus, K.A., Wachsstock, L., Bleeke, J., Krishnan, H.B., Jez, J.M. 2009. Threonine-Insensitive Homoserine Dehydrogenase From Soybean: Genomic Organization, Kinetic Mechanism, and In vivo Activity. Journal of Biological Chemistry. 285(2):827-834.

Interpretive Summary: Soybean is a rich source of protein. Unfortunately, soybean proteins contain low amounts of certain essential amino acid that are vital for optimal growth of humans and animals. Therefore, attempts are being made to increase the amount of these essential amino acids in soybean proteins. In plants the synthesis of four essential amino acids (lysine, threonine, methionine, and isoleucine) occurs through the aspartate amino acid pathway. Homoserine dehydrogenase (HSD) is a critical regulatory enzyme in this pathway. Here we describe the first in-depth biochemical characterization of HSD from soybean. The information obtained from this biochemical study will help biotechnologists to genetically manipulate the aspartate amino acid pathway so that we can improve the overall quality of soybean seed proteins. Superior quality soy proteins can be utilized to meet the nutritional requirements of the multitude of malnourished people around the world as well as improve the quality of the animal feed.

Technical Abstract: Aspartate kinase (AK) and homoserine dehydrogenase (HSD) functions as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback inhibited by threonine. In plants, the biochemical properties of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular functions of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding a monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies demonstrate that the enzyme uses 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 (Ki=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 (Ki~150 micro-molar). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant and -sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first isolation and molecular 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.