Cellular and Molecular Life Sciences

, Volume 71, Issue 5, pp 933–948

Acyl-CoA thioesterase 9 (ACOT9) in mouse may provide a novel link between fatty acid and amino acid metabolism in mitochondria

  • Veronika Tillander
  • Elisabet Arvidsson Nordström
  • Jenny Reilly
  • Malgorzata Strozyk
  • Paul P. Van Veldhoven
  • Mary C. Hunt
  • Stefan E. H. Alexson
Research Article

DOI: 10.1007/s00018-013-1422-1

Cite this article as:
Tillander, V., Arvidsson Nordström, E., Reilly, J. et al. Cell. Mol. Life Sci. (2014) 71: 933. doi:10.1007/s00018-013-1422-1

Abstract

Acyl-CoA thioesterase (ACOT) activities are found in prokaryotes and in several compartments of eukaryotes where they hydrolyze a wide range of acyl-CoA substrates and thereby regulate intracellular acyl-CoA/CoA/fatty acid levels. ACOT9 is a mitochondrial ACOT with homologous genes found from bacteria to humans and in this study we have carried out an in-depth kinetic characterization of ACOT9 to determine its possible physiological function. ACOT9 showed unusual kinetic properties with activity peaks for short-, medium-, and saturated long-chain acyl-CoAs with highest Vmax with propionyl-CoA and (iso) butyryl-CoA while Kcat/Km was highest with saturated long-chain acyl-CoAs. Further characterization of the short-chain acyl-CoA activity revealed that ACOT9 also hydrolyzes a number of short-chain acyl-CoAs and short-chain methyl-branched CoA esters that suggest a role for ACOT9 in regulation also of amino acid metabolism. In spite of markedly different Kms, ACOT9 can hydrolyze both short- and long-chain acyl-CoAs simultaneously, indicating that ACOT9 may provide a novel regulatory link between fatty acid and amino acid metabolism in mitochondria. Based on similar acyl-CoA chain-length specificities of recombinant ACOT9 and ACOT activity in mouse brown adipose tissue and kidney mitochondria, we conclude that ACOT9 is the major mitochondrial ACOT hydrolyzing saturated C2-C20-CoA in these tissues. Finally, ACOT9 activity is strongly regulated by NADH and CoA, suggesting that mitochondrial metabolic state regulates the function of ACOT9.

Keywords

Acyl-CoA thioesterase Propionyl-CoA Amino acid metabolism Fatty acid metabolism Mitochondrial metabolism Organic acids 

Supplementary material

18_2013_1422_MOESM1_ESM.pptx (227 kb)
4,8-Dimethylnonanoyl-CoA is a substrate for ACOT9. 4,8-Dimethylnonanoyl-CoA (DMN-CoA) is formed in peroxisomes via α- and subsequent β-oxidation of phytanic acid. DMN-CoA is a substrate either for carnitine octanoyltransferase (CROT) or ACOT8, a thioesterase with very broad acyl-CoA specificity [66, 67]. DMN can be transferred to mitochondria either as the non-esterified acid or as a carnitine ester, where it can be metabolized to propionyl-CoA and acetyl-CoA via mitochondrial β-oxidation (indicated in the upper right panel). The upper left panel shows a plot of ACOT9 Vmax and Km values with nonanoyl-CoA being clearly distinguishable from the other acyl-CoA esters in the curve. The lower left panel shows a graph of Kcat/Km values including the Kcat/Km for DMN-CoA. The lower right panels show the Michaelis–Menten kinetics of ACOT9 with nonanoyl-CoA (C9-CoA) and DMN-CoA (one representative experiment out of two). Note that Vmax is higher and Km is lower with DMN-CoA, resulting in a 3–4fold higher Kcat/Km (PPTX 226 kb)
18_2013_1422_MOESM2_ESM.pptx (358 kb)
Phylogenetic tree of ACOT9 homologs from selected species ranging from bacteria to primates. The mouse ACOT9 protein sequence was used as a template to search various databases and full-length protein sequences were aligned using ClustalW. While some species contain more than one ACOT9 homologue, e.g. C. elegans and Danio rerio, some mammalian species including human and other primates contain one gene that is alternatively spliced (3rd exon coding for nine amino acids) resulting for proteins of 439 and 448 amino acids, indicated as -1 and -2 respectively. *Indicates the presence of putative C-terminal peroxisomal Type-1 targeting signal, with its amino acid sequence being added after the name (PPTX 358 kb)
18_2013_1422_MOESM3_ESM.pptx (437 kb)
Identification of the putative catalytic residues and active sites I and II in ACOT9. The protein sequences of mouse ACOT7, ACOT9, ACOT11, ACOT12 and ACOT13 were aligned together with a homologous thioesterase from H. walsbyi (Haloquadratum walsbyi) using ClustalW. The upper panel (a cladogram) shows the relationship of H. walsbyi thioesterase, ACOT7, ACOT9, ACOT11, ACOT12 and ACOT13 despite low sequence similarity. It should be noted that ClustalW uses the ‘neighbor-joining method’, which rather represent the data in the form of an additive tree and therefore provides a branching pattern rather than relative time or relative amount of amino acid changes (evolutionary changes). The lower panel shows the alignment of the corresponding amino acid sequences that highlights (boxed) the catalytic residues of active site I (Asn-36 and Asp-225, which are crucial for activity) and II (Glu-51 and Thr-210) of ACOT7 as identified in [15]. In ACOT9, ACOT11 and ACOT12 only site II is active (corresponding Asp and Asn residues). ACOT13 and the H. walsbyi thioesterase contain only one thioesterase domain with one active site (Asn and Asp) that upon dimerization of the proteins results in two active sites (Asn/Asp and Asn/Asp respectively) per dimer in these proteins (PPTX 436 kb)
18_2013_1422_MOESM4_ESM.pptx (812 kb)
The catalytic residues of the putative active site II (Asp and Asn) are conserved in a variety of ACOT9 homologs. A selection of ACOT9 homologs from various species from archaea bacteria to human were aligned using ClustalW. Note that Asp and Asn of active site II are conserved in all ACOT9 proteins containing two thioesterase domains. The archaea bacterium Haloquadratum walsbyi (H. walsbyi) is included as a bacterial homologue containing only one thioesterase domain and Desulfobulbus propionicus (D. propionicus) and Geobacter uraniireducens (G. uraniireducens) are included as examples of bacterial ACOT9 homologs containing two thioesterase domains. M. musculus; Mus musculus, C. elegans; Caenorhabditis elegans, C. intestinalis; Ciona intestinalis, H. sapiens, Homo sapiens, I. scapularis; Ixodes scapularis, M. gallopavo; Meleagris gallopavo, N. vectensis; Nematostella vectensis, R. communis, Ricinus communis, T. stipitatus; Talaromyces stipitatus, V. carteri; Volvox carteri (PPTX 812 kb)

Copyright information

© Springer Basel 2013

Authors and Affiliations

  • Veronika Tillander
    • 1
  • Elisabet Arvidsson Nordström
    • 1
  • Jenny Reilly
    • 1
  • Malgorzata Strozyk
    • 1
  • Paul P. Van Veldhoven
    • 2
  • Mary C. Hunt
    • 3
  • Stefan E. H. Alexson
    • 1
  1. 1.Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska InstitutetC1-74, Karolinska University HospitalStockholmSweden
  2. 2.Department of Cellular and Molecular Medicine, LIPIT, Campus GasthuisbergKatholieke Universiteit LeuvenLeuvenBelgium
  3. 3.Dublin Institute of TechnologySchool of Biological SciencesDublin 8Ireland

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