Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

Plant Mol Biol. 1994 Oct;26(2):631-42. doi: 10.1007/BF00013749.

Abstract

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The 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 band of 1500 bp that after sequencing showed an open reading frame of 382 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 fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison 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 sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Chloroplasts / enzymology
  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • Cloning, Molecular / methods
  • DNA Primers
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / isolation & purification*
  • Electrophoresis, Polyacrylamide Gel
  • Fatty Acid Desaturases / biosynthesis
  • Fatty Acid Desaturases / isolation & purification*
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Phylogeny
  • Plant Leaves / enzymology
  • Plastids / enzymology*
  • Polymerase Chain Reaction / methods
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / metabolism
  • Sequence Homology, Amino Acid
  • Spinacia oleracea / enzymology*
  • Spinacia oleracea / genetics

Substances

  • DNA Primers
  • DNA, Complementary
  • RNA, Messenger
  • Fatty Acid Desaturases
  • delta-12 fatty acid desaturase

Associated data

  • GENBANK/X78311