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) and 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) from the cDNA expression library of Canavalia lineata leaves constructed in a lambdaZAP Express vector. ClOCT1 and ClOCT2 encode 359 and 369 amino acids, respectively. The N-terminals of deduced amino acid sequences of the two cDNAs showed typical features of the transit peptide of chloroplast 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 and Arabidopsis thaliana. However, the CP-binding site sequence of ClOCT2, SLRTH, has not yet been reported. Both ClOCT1 and ClOCT2 cDNAs were expressed in Escherichia coli BL21 (DE3) by using expression vector pET30a. Recombinant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity. In contrast, recombinant ClOCT2 protein showed 13 times higher canaline-dependent OCT activity than ornithine-dependent OCT activity. The two amino acids of the CP-binding site of ClOCT2 (SLRTH) were combinatorially changed to those of the CP-binding site of ClOCT1 (SMRTR) by site-directed mutagenesis. When Leu-118 of ClOCT2 was changed to Met, ornithine-dependent activity was increased significantly. It is assumed that the substrate specificity of ClOCT1 or ClOCT2 proteins partially depends on the amino acid sequence of the CP-binding site.
Lee
Y
Y
School of Biological Sciences, Seoul National University, Korea.
Choi
Y A
YA
Hwang
I D
ID
Kim
S G
SG
Kwon
Y M
YM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019685:Rosales / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D020033:Protein Isoforms; D002221:Carbamyl Phosphate; D009954:Ornithine Carbamoyltransferase