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J. Biol. Chem., Vol. 278, Issue 31, 29016-29023, August 1, 2003

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

From the ^aUnit of Biochemistry and Molecular Biology, International University of Catalonia, 08190 Sant Cugat del Vallés, ^bBioinformatics Laboratory (CAB-CSIC), 28850 Torrejón de Ardoz, Madrid, ^cDepartment of Pharmacology and Physiology, University of Zaragoza, 50009 Zaragoza, ^eProtein Design Group (CNB-CSIC), Cantoblanco, 28049 Madrid, the ^gDepartment of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain, ^hWillink Biochemical Genetic Unit, Manchester M27 4HA, United Kingdom, the ⁱDepartment of Pediatrics, Leicester General Hospital, Leicester LE5 4PW, United Kingdom, ^jFundación para el Estudio de las Enfermedades Neurometabólicas, 1425 Buenos Aires, Argentina, and ^kDivision of Metabolic and Endocrine Diseases, University Children's Hospital, D-69120 Heidelberg, Germany

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 ()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 -sheets around the substrate cavity.

Received for publication, April 23, 2003 , and in revised form, May 13, 2003.

^* This work was supported in part by Grant PB95-0012 from the Dirección General de Investigación Científica y Técnica, the Fundació de la Marató de TV3 (Barcelona), Grant G03/054 from the Spanish Ministry of Health, Grant 2001SGR0123 from the Generalitat de Catalunya, Spain (to F. G. H.), Grant CICYT 2002-2003 (to A. V.), a grant from the Ramon Areces Foundation (to P. G.-P.), and Grant P100/99-BM from the Diputación General de Aragón (to J. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^d Recipient of a fellowship from the Universitat Internacional de Catalunya.

^f Recipient of fellowship from the Diputación General de Aragón.

^l Present address: Dept. of General Pediatrics, University Children's Hospital, Heinrich-Heine-University Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany.

^m To whom correspondence should be addressed: Dept. of Biochemistry, School of Pharmacy, Avda. Diagonal 643, E-08028 Barcelona, Spain. Tel.: 34-93-402-4523; Fax: 34-93-402-4520; E-mail: hegardt{at}farmacia.far.ub.es.

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