cDNA cloning of two isoforms of ornithine carbamoyltransferase from Canavalia lineata leaves and the effect of site-directed mutagenesis of the carbamoyl phosphate binding site

The immunoscreening method was used to isolate cDNAs of 1323 bp (ClOCT1) an d 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2. 1.3.3) from the cDNA expression library of Canavalia lineata leaves constru cted in a lambda ZAP Express vector. ClOCT1 and ClOCT2 encode 359 and 369 a mino acids, respectively. The N-terminals of deduced amino acid sequences o f the two cDNAs showed typical features of the transit peptide of chloropla st targeting proteins. The ornithine-binding domain (FMHCLP) and catalytic domain (HPXQ) of ClOCT1 and ClOCT2 and the carbamoyl phosphate (CP)-binding site of ClOCT1 (SMRTR) are identical to OCTs of other plant species, pea a nd Arabidopsis thaliana. However, the CP-binding site sequence of ClOCT2, S LRTH, has not yet been reported. Both ClOCT1 and ClOCT2 cDNAs were expresse d in Escherichia coli BL21 (DE3) by using expression vector pET30a. Recombi nant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity. In contrast, recombinant ClOCT2 prot ein showed 13 times higher canaline-dependent OCT activity than ornithine-d ependent OCT activity. The two amino acids of the CP-binding site of ClOCT2 (S (S) under bar RT (H) under bar) were combinatorially changed to those o f the CP-binding site of ClOCT1 (S (M) under bar RT (R) under bar) by site- directed mutagenesis. When Leu-118 of ClOCT2 was changed to Met, ornithine- dependent activity was increased significantly. It is assumed that the subs trate specificity of ClOCT1 or ClOCT2 proteins partially depends on the ami no acid sequence of the CP-binding site.