Inhibition of Threonine Dehydratase Is Herbicidal.

Threonine dehydratase, the first enzyme in isoleucine biosynthesis, catalyzes deamination and dehydration of threonine to produce 2-ketobutyrate and ammonia. An antimetabolite, 2-(1-cyclohexen-3(R)-yl)-S-glycine (CHG), inhibits the plant enzyme. CHG inhibits the growth of Black Mexican Sweet corn (Zea mays) cells and of Arabidopsis thaliana plants. The herbicidal effects of CHG can be reversed by 2-ketobutyrate, other intermediates of isoleucine biosynthesis, and by isoleucine itself. These results suggest that the herbicidal effects observed with CHG are a consequence of inhibition of threonine dehydratase. The enzyme could be a potential target site for an herbicide screening program.

Identification and Characterization of a Biodegradative Form of Threonine Dehydratase in Senescing Tomato (Lycopersicon esculentum) Leaf.

Threonine dehydratase (TD; EC.4.2.1.16) is a key enzyme involved in the biosynthesis of isoleucine. Inhibition of TD by isoleucine regulates the flow of carbon to isoleucine. We have identified two different forms of TD in tomato (Lycopersicon esculentum) leaves. One form, present predominantly in younger leaves, is inhibited by isoleucine. The other form of TD, present primarily in older leaves, is insensitive to inhibition by isoleucine. Expression of the latter enzyme increases as the leaf ages and the highest enzyme activity is present in the old, chlorotic leaves. The specific activity of the enzyme present in older leaves is much higher than the one present in younger leaves. Both forms can use threonine and serine as substrates. Whereas TD from the older leaves had the same Km (0.25 mM) for both substrates, the enzyme from the young leaves preferred threonine (Km = 0.25 mM) over serine (Km = 1.7 mM). The molecular masses of TD from the young and the old leaves were 370,000 and 200,000 D, respectively. High levels of the isoleucine-insensitive form of threonine dehydratase in the older leaves suggests an important role of threonine dehydratase in nitrogen remobilization in senescing leaves.

A high-performance liquid chromatography assay for threonine/serine dehydratase.

Threonine dehydratase, a key enzyme leading to the biosynthesis of isoleucine, catalyzes the production of 2-ketobutyrate from threonine. An uv/fluorometric HPLC assay for threonine dehydratase has been developed that involves derivatization of the 2-ketoacids produced by the enzyme using a specific derivatizing agent, o-phenylenediamine. The derivatized ketoacids can be detected spectrophotometrically or fluorometrically. This novel assay is over 1000-fold more sensitive than the commonly used dinitrophenyl hydrazine derivatization assay. In addition, the HPLC assay allows the identification of the reaction product(s), and can be used to examine the reaction using mixtures of the two substrates, threonine and serine.

Arabidopsis Acetohydroxyacid Synthase Expressed in Escherichia coli Is Insensitive to the Feedback Inhibitors.

Acetohydroxyacid synthase (AHAS), the first enzyme unique to the biosynthesis of isoleucine, leucine, and valine, is the target enzyme for several classes of herbicides. The AHAS gene from Arabidopsis thaliana, including the chloroplast transit peptide, was cloned into the bacterial expression plasmid pKK233-2. The resulting plasmid was used to transform an AHAS-deficient Escherichia coli strain MF2000. The growth of the MF2000 strain of E. coli was complemented by the functional expression of the Arabidopsis AHAS. The AHAS protein was processed to a molecular mass of 65 kilodaltons that was similar to the mature protein isolated from Arabidopsis seedlings. The AHAS activity extracted from the transformed E. coli cells was inhibited by imidazolinone and sulfonylurea herbicides. AHAS activity extracted from Arabidopsis is inhibited by valine and leucine; however, this activity was insensitive to these feedback inhibitors when extracted from the transformed E. coli.