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

Resultado de la investigación: Contribución a una revistaArtículorevisión exhaustiva

25 Citas (Scopus)

Resumen

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.

Idioma originalInglés
Páginas (desde-hasta)235-243
Número de páginas9
PublicaciónMolecular Genetics and Metabolism
Volumen75
N.º3
DOI
EstadoPublicada - 2002
Publicado de forma externa

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