Acyl-acyl carrier protein synthetases from bluegreen algae and plants

Abstract

All metabolic processes involving fatty acids are preceded by the activation of the fatty acid to form a thioester derivative. Activation by acyl-CoA synthetase is well established whereas only little information is available about the alternative way utilizing acyl carrier protein (ACP) as acceptor of the acyl group. The focus of this work was to investigate acyl-ACP synthetase (AAS) activity in cyanobacteria and plants, two groups of photosynthetic organisms.In a previous study of our group cyanobacterial AAS was identified and functionally characterized by heterologous expression in Escherichia coli. However, the biological role of this activity within the cell was not determined. Two homologous sequences, AAE15 and AAE16 were found in Arabidopsis. Both of them were investigated by Koo et al. (2005) with respect to their role in the elongation of exogenous fatty acids in chloroplasts, but only for AAE15 the AAS activity was proposed.To elucidate the biological role of AAS activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. The obtained mutants showed two phenotypes: They were unable to utilize exogenous fatty acids and they secreted endogenous fatty acids into the culture medium. The wild type phenotype was restored by complementation of the aas knockout mutant of Synechocystis sp. PCC 6803 with the corresponding gene from Synechococcus elongatus PCC 7942. The analysis of extracellular and intracellular fatty acid profiles of wild type strains and aas mutant strains showed that fatty acids are released from membrane lipids indicating a strong turnover of these lipids. The AAS activity seemed to be necessary for the reactivation of the released fatty acid thereby enabling their recycling back into the lipid metabolism.The genome of Arabidopsis encodes two sequences with strong amino acid similarity to the AAS of cyanobacterial origin. For these two proteins termed AAE15 and AAE16 the localization in plastids was confirmed by expression of EYFP fusion proteins in onion cells followed by fluorescent microscopy. To further characterize these proteins, AAE15 and AAE16 were overexpressed in insect cells and analyzed by in-vitro assays. It was shown that AAE15 displays AAS activity with substrate specificity towards medium chain fatty acids. In addition the aas knockout mutant of Synechocystis sp. PCC 6803 was complemented individually with AAE15 and AAE16, and the obtained strains were fed with labeled fatty acids. It was demonstrated that complementation with AAE15 restored the wild type phenotype, and the specificity for medium chain fatty acids of the enzyme was confirmed. In addition the complementation experiment gave evidence for AAS activity of AAE16 and indicated its broad substrate specificity. In further studies the expression of AAE15 and AAE16 were analyzed with respect to tissue specificity and to dependence on developmental stage. The histochemical GUS staining indicated highly specific expression profiles for AAE15 and AAE16 in flower organs and additionally for AAE16 in guard cells of different organs. The results revealed some strong discrepancies to microarray-based data provided by Genevestigator.