Cloning and Sequencing of Chickpea cDNA Coding for Threonine Deaminase

TD (EC 4.2.1.16) is the enzyme catalyzing the conversion of L-Thr to a-ketobutyrate, the first step of the pathway leading to the biosynthesis of the essential amino acid, L-Ile. Regulation of TD activity by Ile was the first recognized instance of allosteric feedback regulation by the end product of a biosynthetic pathway (Umbarger, 1956). The substrate L-Thr is a feedback inhibitor of aspartokinase and homoserine dehydrogenase (Umbarger, 1978; Jones and Fink, 1982), two of the key regulatory enzymes of the branched chain amino acid pathway. The role of TD in plant cell proliferation and differentiation influenced by Ile, Leu, and Met has been reported earlier from our laboratory (Basu et al., 1989). To extrapolate these biochemical data to the molecular level, we attempted to isolate cDNA for TD. Here we report the cloning and complete nucleotide sequence of TD cDNA from Cicev av ie t inum L. and expression of TD transcript in different parts of the plant. The TD genes from Eschevickia coli ilv A (Cox et al., 1987), yeast ilvl (Kielland-Brandt et al., 1984), and tomato (Samach et al., 1991) have been cloned and characterized. Homologous domains conserved ín evolution, denoted as C 1-5 and R 1-7, have been identified and characterized (Taillon et al., 1988). The 1.1-kb Psfl fragment of pC519 containing ilvl gene (Kielland-Brandt et al., 1984) from yeast was used as a probe to isolate the chickpea homolog. The cDNA library was constructed in A Zap I1 using poly(A)+ RNA from immature seeds of chickpea. Severa1 rounds of plaque hybridization were carried out using the probe to isolate the homologous clone. The clone for Cicer TD cDNA, pCiTD2, is 1872 bp long and its deduced polypeptide has 590 amino acids. The N terminus of the deduced polypeptide contains a typical two-domain transit peptide consistent with chloroplast lumen targeting sequences (Keegstra et al., 1989), indicating chloroplast localization of the mature protein (Table I). The N-terminal domain (45 residues) is rich in Thr and Ser (37%), and the rest of the sequence (46