Cloning, expression and purification of an acetoacetyl CoA thiolase from sunflower cotyledon

Abstract

Thiolase I and II coexist as part of the glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT.

Keywords: acetoacetyl CoA; cloning; expression; sunflower; thiolase.

Figure 1

Enzymatic activities of Thiolase I and Thiolase II. Acetyl CoA produced by Thiolase I (EC 2.3.1.16, 3-ketoacyl-CoA thiolase KAT) activity is used in the TCA cycle for energy production, while acetoacetyl CoA resulting from Thiolase II (EC 2.3.1.9, acetoacetyl CoA thiolase AACT) activity is used in anabolic processes including ketone body and sterol synthesis.

Figure 2

Primer design for 3'-RACE. The conserved domain database (CCD) search tool was used to obtain the consensus sequence for the C-terminal domain of thiolases. This consensus sequence was backtranslated to DNA (C-termCD: C-terminal conserved domain) and a BLAST search was performed with the resulting sequence. A. thaliana sequence (NM_203172) was chosen as template for the primer design and the highlighted sequence was synthesized as 3'-RACE primer Athal.

Figure 3

Alignment of 3'-RACE PCR product with A. thaliana thiolase (NM_203172). The putative sunflower AACT shares 70% sequence identity with the Arabidopsis sequence. Reverse complements of the underlined bold sequences were the primer sequences Sun1 and Sun2, which were the 3'-primers for the 5'-RACE protocol.

Figure 4

Nucleotide and deduced amino acid sequences of the AACT (thiolase II) from sunflower (Helianthus annuus L.) cotyledon. The sequences have been submitted to GenBank (Accession No. GQ254017).

Figure 5

Coomassie blue-stained SDS-PAGE and thiolase activity assay of recombinant sunflower AACT. (A) Recombinant thiolase expressed in E. coli and purified with nickel-affinity chromatography. Equal volumes of each fraction were prepared and resolved on a 0.75 mm 4% stacking/10% resolving minigel (BioRad). Lane 1: Lysate supernatant; Lane 2: flow-through from column; Lanes 3, 5-9: elution fractions (10, 50, 100, 125, 150, 250 mM imidazole); lane 4: Invitrogen Mark 12 standards , kDa. Arrow at 47 kDa indicates the AACT.

Figure 6

Multiple sequence alignment of the closest hits of a BLAST search performed with the deduced amino acid sequence of Helianthus annuus AACT showing the C-termini only. Alignment was done using CLUSTAL W2. The accession numbers are as follows: AAU95619 (N. tabacum), XP_002522876 (R. communis), AF429383_1 (H. brasiliensis), BAF98276 (H. brasiliensis 2), ABC74567 (Picrorhiza kurrooa), BAD22334 (Oryza sativa Japonica Group), NP_199583 (A. thaliana), XP_002308755 (P. trichocarpa), ACN40771 (P. sitchensis), ACF85124 (Z. mays), XP_002320528 (P. trichocarpa 2), XP_002265690 V. vinifera), EEC69797 (O. sativa Indica Group).