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Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure.
Mol Genet Metab 2002; 75(3):235-43MG

Abstract

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 degrees C. Expression of the Q145E mutant cDNA resulted in about 12.5% normal amount at 37 degrees 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 degrees C and one-half normal at 30 degrees C for A127V, and 25 % only at 30 degrees C for G152A. Mutant proteins with Q145E, G152A, or A127V accumulated at 30 degrees C expression were stable for 48 h at 37 degrees 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.

Authors+Show Affiliations

Department of Pediatrics, Gifu University School of Medicine, Gifu, Gifu 500-8076, Japan. toshi-gif@umin.ac.jpNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Case Reports
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11914035

Citation

Fukao, Toshiyuki, et al. "Characterization of Six Mutations in Five Spanish Patients With Mitochondrial acetoacetyl-CoA Thiolase Deficiency: Effects of Amino Acid Substitutions On Tertiary Structure." Molecular Genetics and Metabolism, vol. 75, no. 3, 2002, pp. 235-43.
Fukao T, Nakamura H, Nakamura K, et al. Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure. Mol Genet Metab. 2002;75(3):235-43.
Fukao, T., Nakamura, H., Nakamura, K., Perez-Cerda, C., Baldellou, A., Barrionuevo, C. R., ... Kondo, N. (2002). Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure. Molecular Genetics and Metabolism, 75(3), pp. 235-43.
Fukao T, et al. Characterization of Six Mutations in Five Spanish Patients With Mitochondrial acetoacetyl-CoA Thiolase Deficiency: Effects of Amino Acid Substitutions On Tertiary Structure. Mol Genet Metab. 2002;75(3):235-43. PubMed PMID: 11914035.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure. AU - Fukao,Toshiyuki, AU - Nakamura,Haruki, AU - Nakamura,Kozue, AU - Perez-Cerda,Celia, AU - Baldellou,Antonio, AU - Barrionuevo,Carlos R, AU - Castello,Francisco Girona, AU - Kohno,Yoshinori, AU - Ugarte,Magdalena, AU - Kondo,Naomi, PY - 2002/3/27/pubmed PY - 2002/6/19/medline PY - 2002/3/27/entrez SP - 235 EP - 43 JF - Molecular genetics and metabolism JO - Mol. Genet. Metab. VL - 75 IS - 3 N2 - 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 degrees C. Expression of the Q145E mutant cDNA resulted in about 12.5% normal amount at 37 degrees 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 degrees C and one-half normal at 30 degrees C for A127V, and 25 % only at 30 degrees C for G152A. Mutant proteins with Q145E, G152A, or A127V accumulated at 30 degrees C expression were stable for 48 h at 37 degrees 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. SN - 1096-7192 UR - https://www.unboundmedicine.com/medline/citation/11914035/Characterization_of_six_mutations_in_five_Spanish_patients_with_mitochondrial_acetoacetyl_CoA_thiolase_deficiency:_effects_of_amino_acid_substitutions_on_tertiary_structure_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096719201932883 DB - PRIME DP - Unbound Medicine ER -