Structural (βα)8 TIM Barrel Model of 3-Hydroxy-3-methylglutaryl-Coenzyme A Lyase

Núria Casals,José E Abdenur,Juan Pié,Jacqueline Till,Fausto G Hegardt,Alfonso Valencia,Juan C Cresto,Rosa Aledo,Cecilia Mir,Guillermina Asins,Beatriz Puisac,Josep Clotet,Sebastián Menao,Ramón Roca,Ertan Mayatepek,Dolors Serra,A C Elias-Jones ,G T N Besley ,N Chamoles ,Paulino Gómez‐Puertas

doi:10.1074/jbc.m304276200

Abstract

This study describes three novel homozygous missense mutations (S75R, S201Y, and D204N) in the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase gene, which caused 3-hydroxy-3-methylglutaric aciduria in patients from Germany, England, and Argentina. Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the HMG-CoA lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type. We also propose a three-dimensional model for human HMG-CoA lyase containing a (betaalpha)8 (TIM) barrel structure. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. We also report the localization of all missense mutations reported to date and show that these mutations are located in the beta-sheets around the substrate cavity.

Highlights

This study describes three novel homozygous mis- year of life
Expression studies in Escherichia coli show that S75R and S201Y substitutions completely abolished the hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase activity, whereas D204N reduced catalytic efficiency to 6.6% of the wild type
The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site

Summary

Introduction

This study describes three novel homozygous mis- year of life. Acute episodes include vomiting, lethargy, hypotosense mutations (S75R, S201Y, and D204N) in the 3-hy- nia, and apnea, sometimes evolving to coma [1, 2]. The model is supported by the similarity with analogous TIM barrel structures of functionally related proteins, by the localization of catalytic amino acids at the active site, and by the coincidence between the shape of the substrate (HMG-CoA) and the predicted inner cavity. The three novel mutations explain the lack of HMG-CoA lyase activity on the basis of the proposed structure: in S75R and S201Y because the new amino acid residues occlude the substrate cavity, and in D204N because the mutation alters the electrochemical environment of the active site. The origin of the disease is a mutation in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase (HL), which cleaves HMG-CoA to form acetyl-CoA and acetoacetate. This is the final step in the ketogenic pathway and leucine catabolism. We report three novel homozygous missense mutations affecting patients from Germany, England, and Argentina, and d Recipient of a fellowship from the Universitat Internacional de Catalunya

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Results

Conclusion