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

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 threedimensional 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.3-Hydroxy-3-methylglutaric aciduria (MIM246450) is a rare autosomal recessive metabolic disorder appearing in the 1st year of life. Acute episodes include vomiting, lethargy, hypotonia, and apnea, sometimes evolving to coma (1, 2). Laboratory tests reveal metabolic acidosis with severe hypoketotic hypoglycemia on fasting or during acute illness, hyperammonemia, and abnormal liver function tests. The disease is fatal in about 20% of cases (3) although the symptoms are milder after childhood. Preliminary diagnosis is based on the excretory pattern of organic acids in urine, which include 3-hydroxy-3-methylglutaric, 3-hydroxyisovaleric, 3-methylglutaconic, 3-methylglutaric, and 3-methylcrotonic acids (1, 4).

Crystal Structure of Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

“…However, it is not obvious how changes caused by this mutation are structurally communicated into the active site. Because D204N could also make the same hydrogen bonds and no role for the negative charge of Asp-204 is apparent in the structure, it is more puzzling that D204N has diminished activity (14% of wild type V m (15)). Glu 279 is found on ␣-helix 10, which is not part of the (␤␣) 8 barrel.…”

“…3B, left), although the C-terminal face is accessible to the active site cavity (Fig. 3B, right) (15). In malate synthase, a TIM barrel enzyme that catalyzes a homologous Claisen reaction, the acetyl-CoA-binding site is on the C-terminal side of the barrel (31).…”

“…Recently, a three-dimensional model proposing a substrate binding cavity for human HMG-CoA lyase has been reported (15). This model is interpreted to suggest that HMG-CoA binds through a central pore of the (␤␣) 8 barrel of the enzyme.…”

“…Two serine mutations, S75R and S201Y, have been identified, and the recombinant proteins were expressed (15). Neither of these enzymes was found to be active in the soluble fraction of crude extracts.…”

Crystal Structures of Two Bacterial 3-Hydroxy-3-methylglutaryl-CoA Lyases Suggest a Common Catalytic Mechanism among a Family of TIM Barrel Metalloenzymes Cleaving Carbon-Carbon Bonds

“…A homology model for the Michaelis complex of the enzyme has been published based on the structure of HisA (PDB code 1QO2) (14,15). In this model, the lipophilic moiety of the CoA passes through the core of the TIM barrel (i.e.…”