It is estimated that approximately 4% of enzymes use CoA as a cofactor or act on CoA-linked substrates. In plants, pathways of fatty acid and lipid synthesis, including isoprenoids, sterols, cutin and suberin, the tricarboxylic acid cycle, and amino acid synthesis all rely on CoA, as does the breakdown of fatty acids by b-oxidation. Many pathways of secondary product synthesis also use CoA-linked intermediates, including those leading to lignins, lignans, flavonoids and glucosinolates. Many far less-studied pathways also involve CoA metabolism. CoA ligases and some related proteins are characterized by a highly conserved 12-amino-acid sequence that forms a core of the so-called AMP-binding motif (PROSITE PS00455). This feature made it possible to identify a family of 63 genes in Arabidopsis that encode acyl activating enzymes (AAEs), of which 44 are CoA ligases (or functionally related ACP ligases). The identification has a high degree of confidence even though overall sequence identity across the family is less than 25% and falls below 20% when three highly conserved regions are excluded from the analysis. Enzymes that hydrolyze CoA thioesters are less conserved and belong to several unrelated families. Nevertheless, it has been possible to identify 15 genes encoding putative CoA thioesterases, and these will also be important to our analysis of CoA-linked pathways.
In this project, most of the genes we will study have a well-defined general function. Our task will be to discover the precise function of each, and then use this knowledge to illuminate the many pathways to which they collectively contribute. Only 10 of the 44 CoA ligase genes and 1 of the 15 CoA thioesterase genes have well-defined functions. We expect to pinpoint key reactions in a spectrum of different primary and secondary metabolic pathways, including those involved in the synthesis and breakdown of several plant hormones. In cases where a pathway was previously unknown in Arabidopsis, or recalcitrant to investigation, the work described will provide an entry point for further studies. Our investigations of the biochemistry and cell biology of each gene product together with the identification and characterization of the corresponding mutant will supply the means both to sketch proposed pathways, and to test these proposals using biochemical and genetic approaches.
A proportion of the CoA ligase and thioesterase enzymes will likely be found to act in pathways that turnover chemical components of the cell and extracellular matrix. In animals, disruption of such pathways can have striking developmental and physiological consequences. Similarly, in our lacs6/lacs7 double mutant, seedling development is blocked because storage lipids cannot be catabolized rapidly. The characterization of the aim1 mutant of Arabidopsis, which is defective in a specific isoform of the b-oxidation multifunctional protein, also indicates that disruption of degradation pathways can affect multiple aspects of development and physiology.