Molecular cloning of a cDNA encoding 3-ketoacyl-acyl carrier protein synthase III from leek

Abstract

3-Ketoacyl-acyl carrier protein synthase III (KAS III) catalyzes the initial condensation of malonyl-acyl carrier protein (ACP) with acetyl-CoA in plant and bacterial fatty acid biosynthesis. The first cDNA clone encoding KAS III from a monocot is reported here. A cDNA clone was isolated from a leek epidermal cDNA library by screening with spinach and Arabidopsis heterologous probes from KAS III cDNA clones. When expressed in Escherichia coli, the cloned enzyme was able to catalyze the expected condensation reaction, was insensitive to cerulenin (100 μM) and cross-reacted with spinach KAS III antibody. The 1476-bp cDNA clone contained a 1206-bp open reading frame which encoded a 402-amino acid polypeptide. The deduced amino acid sequence showed significant similarities to other KAS IIIs although the leek sequence had some notable differences in regions otherwise completely conserved in dicots. Northern blot analyses indicated that KAS III transcript levels were similar in leaf epidermis and parenchyma, although developmental changes in transcript levels differed between these two tissues. In addition, leek KAS III was expressed in a manner comparable to leek oleoyl-ACP thioesterase (OTE), another enzyme of fatty acid biosynthesis, in both tissues.