PURIFICATION AND PCR-BASED CDNA CLONING OF A PLASTIDIAL N-6 DESATURASE

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oler acea) was purified from chloroplast envelope membranes by anion exchan ge, cation exchange and ferredoxin-affinity chromatography. The molecu lar mass of the protein was estimated by SDS-PAGE to be 40 kDa. The hi ghest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. Th e N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3'-RACE experiment with this primer amplified a single b and of 1500 bp that after sequencing showed an open reading frame of 3 82 amino acids corresponding to a protein of 43 kDa. The 5' end of the cDNA was amplified by a 5'-RACE experiment and isolated as a 500 bp f ragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plast idial leader peptide. With appropriate primers derived from these sequ ences a full-length clone was amplified by PCR and sequenced. Comparis on of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50 % amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequ ence HXXXH that are highly conserved in all membrane-bound desaturases . These boxes might be involved in metal ion complexation required for reduction of oxygen.