Characterisation of the gene family encoding acetoacetyl-CoA thiolase in Arabidopsis

Abstract

Thiolases are ubiquitous enzymes involved in many essential biochemical processes. Biosynthetic thiolases, also known as acetoacetyl-CoA thiolases (AACT), catalyse a reversible Claisen-type condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. Here, we report the characterisation of two genes from Arabidopsis thaliana L., ACT1 and ACT2, which encode two closely related AACT isoforms (AACT1 and AACT2, respectively). Transient expression of constructs encoding AACT1 and AACT2 fused to GFP revealed that the two proteins show a different subcellular localisation. While AACT1 is found in peroxisomes, AACT2 localises in the cytosol and the nucleus. The peroxisomal localisation of AACT1 depends on the presence of a C-terminal peroxisomal targeting sequence (PTS1) motif (Ser-Ala-Leu) not previously found in other organisms. ACT1 and ACT2 genes are also differentially expressed. Whereas ACT2 is expressed at relatively high level in all plant tissues, the expression of ACT1 is restricted to roots and inflorescences and its transcript is present at very low levels. The obtained results are in agreement with the involvement of AACT2 in catalysing the first step of the mevalonate pathway. The metabolic function of AACT1 is not clear at present, although its particular peroxisomal localisation might exclude a role in isoprenoid biosynthesis.

Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

“…The 1233 bp AACT coding sequence is predicted to encode a 411 amino acid protein. This size corresponds well with the length of the other predicted AACTs corresponding to the Arabidopsis annotated gene products from At5g47720 and At5g48230: 415 and 403 amino acids, respectively 17.…”

Overexpression of Brassica juncea wild‐type and mutant HMG‐CoA synthase 1 in Arabidopsis up‐regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance

“…It is likely that the H188N mutation masked the S359A phenotype of increased activity (10‐fold) when expressed in vivo because of the limited affinity to AcAc‐CoA, which is only present in submicromolar concentrations in the cytosol. Moreover, the enzyme preceding HMGS, AcAc‐CoA thiolase favours the thiolytic cleavage of AcAc‐CoA to Ac‐CoA in the presence of HS‐CoA (see Ahumada et al. , 2008).…”

Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta

“…1). Thus, the A. thaliana AACT seems to have dual localisation in both the peroxisome and the cytosol (Carrie et al 2007;Ahumada et al 2008). However, AACT is not restricted to the MVA pathway, and it has been shown that A. thaliana contains two genes AACT1 and AACT2 encoding three and two proteins, respectively.…”