Characterization of six mutations in five spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: Effects of amino acid substitutions on tertiary structure

Toshiyuki Fukao, Haruki Nakamura, Kozue Nakamura, Celia Perez-Cerda, Antonio Baldellou, Carlos R. Barrionuevo, Francisco Girona Castello, Yoshinori Kohno, Magdalena Ugarte, Naomi Kondo

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27 Scopus citations


Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine metabolism. We identified and characterized 6 mutations, DelE85, K124R, A127V, Q145E, G152A, and E345V in 5 Spanish T2-deficient patients. Transient expression of mutant cDNAs was done at 37 and at 30°C. Expression of the Q145E mutant cDNA resulted in about 12.5% normal amount at 37°C and it retained 15% residual T2, indicating that specific activity of Q145E mutant protein was almost normal. This mutation reduced the heat stability of T2 activity. Although no significant residual activity was detected in either the G152A and A127V substitution, mutant proteins were detected, at 12.5% the normal amount at 37°C and one-half normal at 30°C for A127V, and 25% only at 30°C for G152A. Mutant proteins with Q145E, G152A, or A127V accumulated at 30°C expression were stable for 48 h at 37°C after cycloheximide treatment. Expression of DelE85, K124R, and E345V cDNAs gave neither residual T2 protein nor T2 activity. We constructed an improved tertiary structural model of T2 based on the X-ray crystal structure of acetoacetyl-CoA thiolase of Zoogloea ramigera. On the basis of this model, K124, A127, and G152 are located near the active site, mutations of which might affect catalytic function whereas Q145E, De185E, and E345V are distant from the active site with mutants being expected to destabilize the tertiary structure, especially during protein folding and dimerization.

Original languageEnglish
Pages (from-to)235-243
Number of pages9
JournalMolecular Genetics and Metabolism
Issue number3
StatePublished - 2002
Externally publishedYes

Bibliographical note

Funding Information:
We thank Dr. P. Kursula (Department of Biochemistry and Biocenter, Oulu University of Oulu, Finland) for helpful discussion on mutant effects on a teritiary structural model, Dr. L. Sweetman (Institute of Metabolic Disease, Baylor University Medical Center, U.S.A.) for providing GK02 fibroblasts, Professor MF Lavin (Queensland Institute of Medical Research, Brisbane, Australia), and M. Ohara for language assistance and N. Sakagu-chi for technical help. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports, and Culture of Japan.


  • Expression analysis
  • Mitochondrial acetoacetyl-CoA thiolase deficiency
  • Mutations
  • Tertiary structural model
  • β-ketothiolase deficiency


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