Cloning of a cDNA encoding 3-ketoacyl-acyl carrier protein synthase III from Arabidopsis.

The initial step of fatty acid biosynthesis in both plants and Escherichia coli is the condensation of malonyl-acyl carrier protein with acetyl-COA, a reaction that is catalyzed by KAS 111 (Jackowski and Rock, 1987; Jackowski et al., 1989; Jaworski et al., 1989; Clough et al., 1992). In E. coli, KAS 111 has been implicated as a site of regulation for de novo fatty acid synthesis (Jackowski and Rock, 1987; Jackowski et al., 1989). At present, the regulation of fatty acid biosynthesis has not been clearly delineated in plants. Alteration of KAS 111 expression in Arabidopsis by sense and antisense constructs will help elucidate its regulatory role in vivo. The first plant cDNA clone of KAS 111 was recently isolated from spinach (Tai and Jaworski, 1993). The spinach clone showed a high degree of amino acid identity to the E. coli KAS 111 (Tsay et al., 1992) and the putative KAS I11 from Porphyra umbilicalis (red alga) (Reith, 1993), but no similarity to clones of other KAS isozymes from either plants or E. coli. We have isolated an Arabidopsis KAS 111 cDNA clone using the spinach clone as a heterologous probe (Table I). An Arabidopsis cDNA library was screened using moderatestringency conditions with a radiolabeled 612-bp probe generated from the spinach cDNA clone using PCR. Positive X clones were excised into plasmids and rescreened. One of the putative KAS I11 clones showed a strong positive signal in the second screening, and the initial sequencing of the clone revealed a high sequence identity to the 3‘ end of the spinach KAS I11 clone. The full-length clone was sequenced on both strands with customized primers. The Arabidopsis cDNA clone of KAS I11 contains a single open reading frame of 1215 bp encoding a polypeptide that has 71.8% identity to the deduced amino acid sequence of spinach KAS 111. Its active site Cys was identified at position 179 by comparing conserved sequences near the active site of other KAS 111 proteins. The highest degree of homology to spinach KAS 111 was observed near the C tenninus of the peptide (84.9% identity at the residues 300-405), whereas the N tenninus shows more divergence (50% identity at the residues 1-lOO), mainly due to the transit peptide sequence.