An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander

Petroselinic acid (18:1 Delta (6)) is the major component of the seed oil o f Umbelliferae species such as coriander (Coriandrum sativum) as well as Ar aliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the Delta (4) desaturation of palmitoyl-acyl carrier protein (1 6:0-ACP) and subsequent elongation of Delta (4)-hexadecenoyl (16:1 Delta (4 ))-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 Delta (4)-ACP and 16:0-ACP as subst rates. Extracts from developing coriander seeds elongated 16:1 Delta (4)-AC P in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroseli nic acid, displayed a strong preference for the elongation of 16:0-ACP rath er than 16:1 Delta (4)-ACP. In addition, the elongation of 16:1 Delta (4)-A CP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerule nin at concentrations as low as 10 muM. This finding suggested that the elo ngation of 16:1 Delta (4)-ACP and 16:0-ACP in coriander seed is catalyzed b y a 3-ketoacyl-ACP synthase (KAS) I-type enzyme(s), rather than a KAS II-ty pe activity that is typically associated with 16:0-ACP elongation. Consiste nt with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterol ogous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, alt hough the polypeptide corresponding to the coriander cDNA is more closely r elated to KAS I, its active site motif deviates from those found in both KA S I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an a bundant 45 kDa polypeptide in coriander endosperm that is not detected in c oriander leaves. These antibodies also recognize a major band of similar si ze in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.