ACX3, a Novel Medium-Chain Acyl-Coenzyme A Oxidase from Arabidopsis

Abstract

In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0-to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene.

Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes

“…Following substrate import into the peroxisome by PXA1, a CoA moiety is added by one of two long-chain acyl-CoA synthetases (LACS; Fulda et al 2002Fulda et al , 2004. One of six acyl-CoA oxidases (ACX) catalyzes the oxidation step, adding a double bond while releasing hydrogen peroxide (Hayashi et al 1999;Hooks et al 1999;Froman et al 2000;Rylott et al 2003;Adham et al 2005;Pinfield-Wells et al 2005). Next, a multifunctional protein (MFP2 or AIM1), containing both enoyl-CoA hydratase and acyl-CoA dehydrogenase activity, forms a b-ketoacyl-CoA thioester (Richmond and Bleecker 1999;Eastmond and Graham 2000;Rylott et al 2006).…”

Arabidopsis Mutants in Short- and Medium-chain Acyl-CoA Oxidase Activities Accumulate Acyl-CoAs and Reveal That Fatty Acid β-Oxidation Is Essential for Embryo Development

“…The ACX1 gene (Munich Information center for Protein Sequences (MIPS) 1 code At4g16760) is a medium-to longchain ACX with a substrate optimum of C14:0, 2 whereas the ACX2 gene (MIPS code At5g65110) has optimum activity with long-chain saturated and unsaturated acyl-CoAs (C14:0 to C20:0) (13). The ACX3 gene (MIPS code At1g06290/At1g06300) exhibits medium chain length substrate specificity (C8:0-C14:0) (1,14), and AtSCX (ACX4) (MIPS code At3g51840) exhibits short-chain (C4:0-C8:0) substrate specificity (15). Two additional ACX genes, ACX5 (MIPS code At2g35690) and ACX6 (MIPS code At1g06310) are present in the Arabidopsis genome.…”

Gene-Specific Involvement of β-Oxidation in Wound-Activated Responses in Arabidopsis

“…The Arabidopis genome (Arabidopsis Genome Initiative [AGI], 2000), includes six genes encoding ACXs, ACX1 and ACX2 (Hooks et al, 1999); ACX3 (Eastmond et al, 2000;Froman et al, 2000); ACX4 (Hayashi et al, 1999); and the still uncharacterized homologs of ACX1 and ACX3, which we have named ACX1.2, and ACX3.2 (ACX5 and ACX6, respectively, in Rylott et al, 2003); at least two MFPs, AIM1 and MFP2 (Richmond and Bleecker, 1999); and three KATs, PED1/KAT2 (Hayashi et al, 1998;Germain et al, 2001), PKT2/KAT5 (Germain et al, 2001), and KAT1, a homolog of PED1/KAT2 in chromosome 1. Although b-oxidation has been traditionally considered as a catabolic machinery devoted to the production of energy through fatty acid degradation, it can be also considered as a processing system to convert complex precursors into simpler molecules.…”