Malonyl-CoA Synthetase, Encoded by ACYL ACTIVATING ENZYME13, Is Essential for Growth and Development of Arabidopsis      

Abstract

Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (K m = 529.4 ± 98.5 μM; V m = 24.0 ± 2.7 μmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate.

Retracted: Opaque7 Encodes an Acyl-Activating Enzyme-Like Protein That Affects Storage Protein Synthesis in Maize Endosperm

“…One hundred milligrams of dried endosperm flour were used for metabolite extraction according to Chen et al (2011) with some modification. Briefly, dried extracts were dissolved in mixed solution of water/isopropanol/methanol (1:3:6, v/v).…”

A Previously Unknown Oxalyl-CoA Synthetase Is Important for Oxalate Catabolism in Arabidopsis

“…Upon electrophoresis in a SDS-polyacrylamide gel and visualization with Coomassie blue stain, the recombinant protein was estimated to be >90% pure ( Figure 1A). A preparation was tested against 10 carboxylic acids at pH 7.5 and 300 mM substrate, using a spectrophotometric, coupled-enzyme assay (Ziegler et al, 1987;Chen et al, 2011). In these initial assays, His-AAE3 showed high activity only with oxalate, 5.8 mmol/min/mg protein.…”

“…Some CoA synthetase enzymes activate a wide range of substrates in vitro; examples include several long-chain acyl-CoA synthetases that can use multiple fatty acids as substrates (Shockey et al, 2002) and some 4-coumaroyl-CoA ligase-like proteins that were found to use both fatty acids and jasmonate precursors as substrates in vitro (Kienow et al, 2008). By contrast, other AAEs are very specific for their substrate, such as the malonyl-CoA synthetase encoded by AAE13 (Chen et al, 2011). The high specificity of the His-AAE3 recombinant enzyme for oxalate identifies AAE3 as the first gene cloned from any organism to encode oxalyl-CoA synthetase activity.…”

“…Rosette leaves and mature siliques were harvested from plants grown on soil, frozen in liquid nitrogen, and ground. A crude protein extraction method was used to extract the enzyme from the ground tissue as described by Chen et al (2011). Briefly, the frozen ground tissue was added to an extraction buffer solution containing 100 mM Tris-HCl, pH 7.5, 1mM EDTA, 1% (v/v) 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride and centrifuged at 15,500 rcf for 10 min at 48C.…”

Sucrose supplementation suppressed the growth inhibition in polyhydroxyalkanoate-producing plants

“…Mutations in the Arabidopsis malonyl- CoA synthase gene give rise to severe growth inhibition and low fertility (Chen et al 2011); similar effects are observed in ABC-ox, suggesting that growth inhibition in ABC-ox is attributable to low malonyl-CoA. To achieve high PHA yield, we must organize the PHA biosynthetic pathway and endogenous metabolic pathways to avoid interference.…”