In-vitro evidence for feed-back regulation of ?-ketoacyl-acyl carrier protein synthase III in medium-chain fatty acid biosynthesis

Abstract

The cerulenin-insensitive β-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, EC 2.3.1.41) catalyzes the first condensing step of the fatty-acid synthase (FAS) reaction in plants and bacteria, using directly acetyl-CoA as substrate for condensation with malonyl-ACP. In order to identify a possible site for regulation of the biosynthesis of medium-chain fatty acids, the influence of acyl-ACPs of different chain-lengths (C4,C6,C8 and C10) on the activity of KAS III was investigated in vitro using an FAS preparation from seeds of Cuphea lanceolata Ait. (a crop accumulating up to 90% decanoic acid into triacylglycerols) that had been treated with 100 μM cerulenin. All acyl-ACPs investigated led to a decrease in the activity of KAS III towards acetyl-CoA, an effect apparently related to the length of the acyl chain. Analysis of the reaction products of the assay revealed that short-chain acyl-ACPs elongated to a very small extent simultaneously with acetyl-CoA. This extent of elongation did not correlate with the decrease in KAS III-activity levels. These data excluded the possibility of competition between acetyl-CoA and acyl-ACPs, but indicated that acyl-ACPs inhibited the enzyme. Decanoyl-ACP caused the highest decrease in enzyme activity (IC50 = 0.45 μM), thus being a potent inhibitor of KAS III. Michaelis-Menten kinetics revealed that the inhibition of KAS III by decanoyl-ACP was non-competitive in relation to malonyl-ACP and uncompetitive in relation to acetyl-CoA. Moreover, our data indicate that KAS III has a strict specificity for the elongation of acetyl-CoA. An inhibition of KAS III by acyl-ACPs was observed in experiments using FAS preparations from rape seeds and spinach leaves, but the inhibition of KAS III from C. lanceolata seeds by decanoyl-ACP was approximately 1.5-fold higher. The data provide evidence that acyl-ACPs are involved in the modulation of plant fatty-acid biosynthesis by a feed-back mechanism.