Threonine dehydratase (TD) [EC 4.2.1.16] catalyzes the first step in isoleucine biosynthesis. TD deaminates and dehydrates threonine to produce 2-ketobutyrate (2-oxobutyrate or 2-oxobutanoate) and ammonia. A form of this enzyme present predominantly in younger leaves is considered to be the "biosynthetic" form of the enzyme because it is feedback inhibited by isoleucine. The gene encoding the biosynthetic isoenzyme of TD has been isolated from tomato and potato (Singh and Shaner, 1995).

Mutants of Nicotiana plumbaginifolia which are TD-deficient and require isoleucine for growth, have been complemented with the Saccharomyces cerevisiae ilv1 gene encoding TD (Singh and Shaner, 1995).

A second form of TD which is insensitive to inhibition by isoleucine degrades threonine and serine to release ammonia. This isozyme appears in older, senescing leaves, and may be a biodegradative form playing a role in remobilization of the nitrogen of protein amino acids during senescence (Singh and Shaner, 1995).

Acetohydroxyacid synthase (AHAS) [EC 4.1.3.18], also known as acetolactate synthase (ALS), catalyzes the condensation of two molecules of pyruvate to yield acetolactate, and the condensation of pyruvate and 2-oxobutyrate to yield acetohydroxybutyrate.

In both reactions an intermediate is first formed between pyruvate and thiamine pyrophosphate (TPP) as cofactor. This intermediate undergoes decarboxylation to produce a stabilized anion of hydroxyethyl-TPP (HET-PP), which acts as a nucleophile on the 2-keto group of a second molecule of pyruvate or 2-oxobutyrate, releasing TPP and acetolactate or acetohydroxybutyrate. AHAS is dependent on FAD for enzyme activity, although there is no net oxidation or reduction associated with the reaction (Singh and Shaner, 1995).

The number of AHAS genes in plants varies from 1 in Arabidopsis to 5 in Brassica napus (Singh and Shaner, 1995). All appear to contain a putative chloroplast transit peptide sequence, consistent with the localization of AHAS activity in chloroplasts (Singh and Shaner, 1995).

Acetohydroxyacid reductoisomerase [EC 1.1.1.86] or ketol-acid reductoisomerase (KARI) reduces the acetohydroxyacids to produce dihydroxyacids. Only one gene per haploid genome was found in spinach and Arabidopsis. The deduced amino acid sequence contains a transit peptide consistent with a chloroplast localization. The deduced amino acid sequence contains a region characteristic of an NAD(P)H binding site (Singh and Shaner, 1995).

Dihydroxy-acid dehydratase [EC 4.2.1.9] catalyzes the penultimate step in the synthesis of valine and isoleucine. The latter enzyme contains a 2Fe-2S cluster. Nicotiana plumbaginifolia mutants lacking this enzyme require branched-chain amino acids for growth (Singh and Shaner, 1995).

The terminal step in the pathway is catalyzed by an aminotransferase; different isoforms of this enzyme may exist with different specificities for 2-oxoisovalerate, 2-oxo-3-methylvalerate and 2-oxoisocaproate (cf. valine aminotransferase [EC 2.6.1.66], valine-isoleucine aminotransferase [EC 2.6.1.32], and leucine aminotransferase [EC 2.6.1.6]) (Singh and Shaner, 1995).

Bryan JK 1980 Aspartate family and branched-chain amino acids. In (BJ Miflin ed) "The Biochemistry of Plants", Vol. 5, Academic Press, New York, pp. 403-452.

Singh BK, Shaner DL 1995 Biosynthesis of branched chain amino acids: From test tube to field. Plant Cell 7: 935-944.