Arabidopsis contains a large superfamily of acyl-activating enzymes. Phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme a synthetases

Abstract

Acyl-activating enzymes are a diverse group of proteins that catalyze the activation of many different carboxylic acids, primarily through the formation of a thioester bond. This group of enzymes is found in all living organisms and includes the acyl-coenzyme A synthetases, 4-coumarate:coenzyme A ligases, luciferases, and non-ribosomal peptide synthetases. The members of this superfamily share little overall sequence identity, but do contain a 12-amino acid motif common to all enzymes that activate their acid substrates using ATP via an enzyme-bound adenylate intermediate. Arabidopsis possesses an acyl-activating enzyme superfamily containing 63 different genes. In addition to the genes that had been characterized previously, 14 new cDNA clones were isolated as part of this work. The protein sequences were compared phylogenetically and grouped into seven distinct categories. At least four of these categories are plant specific. The tissue-specific expression profiles of some of the genes of unknown function were analyzed and shown to be complex, with a high degree of overlap. Most of the plant-specific genes represent uncharacterized aspects of carboxylic acid metabolism. One such group contains members whose enzymes activate short- and medium-chain fatty acids. Altogether, the results presented here describe the largest acyl-activating enzyme family present in any organism thus far studied at the genomic level and clearly indicate that carboxylic acid activation metabolism in plants is much more complex than previously thought.

Figure 1.

Phylogenetic analysis of the Arabidopsis AAEs. The complete protein sequences of the AAEs were aligned using ClustalX and were displayed graphically using TREEVIEW as described in the text. The branch lengths are proportional to the degree of divergence, with the scale of “0.1” representing 10% change.

Figure 2.

Alignment of the PROSITE PS00455 AMP-binding motif of representative enzymes from each of the seven phylogenetic clades. The enzymes from clade III contain an extra residue relative to the other enzymes, which match the traditional 12-residue consensus sequence.

Figure 3.

Determination of the tissue-specific RNA expression patterns of Arabidopsis AAE genes from clades II (B), VI (C), and VII (A). Gene-specific primer pairs for each gene were used to assess the relative expression levels of each gene by RT-PCR starting from total RNA as described in the “Materials and Methods.” GS, Germinating seedling; R, root; St, stem; L, leaf; F, flower; DS, developing seed; M, M_r marker.

Figure 4.

Biochemical analysis of the short- and medium-chain acyl-CoA synthetase activities expressed from AAE7 (A) and AAE11 (B). Prokaryotic expression constructs were introduced into E. coli and protein overproduction induced with isopropylthio-β-galactoside. Membrane fractions from the induced bacterial cultures were used as enzyme sources in acyl-CoA synthetase assays using radioactive fatty acid substrates. Membranes from E. coli containing the empty vector were used as the negative controls.