A single-residue mutation, G203E, causes 3-hydroxy-3-methylglutaric aciduria by occluding the substrate channel in the 3D structural model of HMG-CoA lyase (original) (raw)
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Structural (betaalpha)8 TIM barrel model of 3-hydroxy-3-methylglutaryl-coenzyme A lyase
The Journal of biological chemistry, 2003
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 residue...
Journal of Biological Chemistry, 2003
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to form acetyl-CoA and acetoacetate. In metaldependent aldol and Claisen reactions, acidic residues often function either as cation ligands or as participants in general acid/base catalysis. Site-directed mutagenesis was used to produce conservative substitutions for the conserved acidic residues Glu-37, Asp-42, Glu-72, Asp-204, Glu-279, and Asp-280. HMG-CoA lyase deficiency results from a human mutation that substitutes lysine for glutamate 279. The E279K mutation has also been engineered; expression in Escherichia coli produces an unstable protein. Substitution of alanine for glutamate 279 produces a protein that is sufficiently stable for isolation and retains substantial catalytic activity. However, thermal inactivation experiments demonstrate that E279A is much less stable than wild-type enzyme. HMG-CoA lyase deficiency also results from mutations at aspartate 42. Substitutions that eliminate a carboxyl group at residue 42 perturb cation binding and substantially lower catalytic efficiency (10 4-10 5-fold decreases in specific activity for D42A, D42G, or D42H versus wildtype). Substitutions of alanine for the other conserved acidic residues indicate the importance of glutamate 72. E72A exhibits a 200-fold decrease in k cat and >10 3-fold decrease in k cat /K m. E72A is also characterized by inflation in the K m for activator cation (26-fold for Mg 2؉ ; >200-fold for Mn 2؉). Similar, but less pronounced, effects are measured for the D204A mutant. E72A and D204A mutant proteins both bind stoichiometric amounts of Mn 2؉ , but D204A exhibits only a 2-fold inflation in K D for Mn 2؉ , whereas E72A exhibits a 12-fold inflation in K D (23 M) in comparison with wild-type enzyme (K D ؍ 1.9 M). Acidic residues corresponding to HMG-CoA lyase Asp-42 and Glu-72 are conserved in the HMG-CoA lyase protein family, which includes proteins that utilize acetyl-CoA in aldol condensations. These related reactions may require an activator cation that binds to the corresponding acidic residues in this protein family.
Mutations underlying 3-Hydroxy-3-Methylglutaryl CoA Lyase
2006
Background: 3-Hydroxy-3-Methylglutaric aciduria (3HMG, McKusick: 246450) is an autosomal recessive branched chain organic aciduria caused by deficiency of the enzyme 3-Hydroxy-3-Methylglutaryl CoA lyase (HL, HMGCL, EC 4.1.3.4). HL is encoded by HMGCL gene and many mutations have been reported. 3HMG is commonly observed in Saudi Arabia. Methods: We utilized Whole Genome Amplification (WGA), PCR and direct sequencing to identify mutations underlying 3HMG in the Saudi population. Two patients from two unrelated families and thirty-four 3HMG positive dried blood spots (DBS) were included. Results: We detected the common missense mutation R41Q in 89% of the tested alleles (64 alleles). 2 alleles carried the frame shift mutation F305fs (-2) and the last two alleles had a novel splice site donor IVS6+1G>A mutation which was confirmed by its absence in more than 100 chromosomes from the normal population. All mutations were present in a homozygous state, reflecting extensive consanguinity. The high frequency of R41Q is consistent with a founder effect. Together the three mutations described account for >94% of the pathogenic mutations underlying 3HMG in Saudi Arabia. Conclusion: Our study provides the most extensive genotype analysis on 3HMG patients from Saudi Arabia. Our findings have direct implications on rapid molecular diagnosis, prenatal and preimplantation diagnosis and population based prevention programs directed towards 3HMG.
Archives of Biochemistry and Biophysics, 1998
Two novel point mutations in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene were found in a French patient with double heterozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by reverse transcriptase-polymerase chain reaction of the mRNA using five different pairs of oligonucleotides produced no differences in the fragments amplified with respect to the control. Single-strand conformation polymorphism analysis showed that only one amplified fragment was different in the patient vs. control. Sequencing of the amplified fragments showed two missense point mutations, A698G and T788C, each of them mixed with the wild-type sequence. These mutations produced the changes H233R and L263P, leading to changes in the enzyme activity, which was largely abolished. The father and one brother of the proband were heterozygous for the L263P mutation and the mother and one daughter were heterozygous for the H233R mutation, which confirms the double-heterozygous character of the patient. Another sibling was free of the mutations. An enzymatic restriction analysis has been proposed to screen the occurrence of these two mutations in future patients.
intechopen.com
The first attempt to build a 3D structural model of human 3-hydroxy-3-methylglutaryl-CoA lyase was based on a threading procedure using the crystallized structure of the TIM-barrel hisA gene from Thermotoga maritima as a template (Casals et al., 2003). The proposed model correspond to a () 8 barrel with short loops on the NH 2 terminal face and, in contrast, long and probably non-structured loops on the COOH-terminal face of the-barrel. This model showed, for the first time, the structural proximity of the residues involved in the cathalytic activity of the protein: Arg 41 , Asp 42 , Glu 72 , His 233 and His 235 , located near the cavity opened in the COOH-terminal face of the protein model (Figure 2). This model was confirmed when the cristal structure of human HL was obtained (Fu et al., 2006). In addition to the basic TIM barrel structure, the monomer of human HL includes an additional polypeptide region made of residues 290-323 containing-strand 9, and-helices 11 and 12. The active site is accessible only from the C-terminal side of the TIM barrel and the N-terminal barrel end is occluded. Crystal structures of the wild-type enzyme complex and inhibitor hydroxyglutaryl-CoA has demostrated the interaction of Arg 41 and acyl-CoA´s C1 carbonyl oxygen of sustrate and explains why Arg 41 mutations cause drastic enzyme deficiency (Fu et al., 2010).
Molecular Genetics and Metabolism, 2007
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase adopts a (ba) 8 TIM barrel structure with an additional b9, a11 and a12 helices. Location of HMG part of the substrate has been suggested but the binding mode for the CoA moiety remains to be resolved. As mutation F305 fs(À2), which involves the last 21 residues of the protein, and mutation K48N caused 3-hydroxy-3-methylglutaric aciduria in two patients, we examined the role of the C-terminal end and Lys 48 in enzyme activity. Expression studies of various C-terminal-enddeleted and K48N-mutated proteins revealed that residues 311-313 (localized in the loop between a11 and a12 helices) and Lys 48 are essential for enzyme activity. An in silico docking model locating HMG-CoA on the surface of the enzyme implicates Asn 311 and Lys 313 in substrate binding by establishing multiple polar contacts with phosphate and ribose groups of adenosine, and Lys 48 by contacting the carboxyl group of the panthotenic acid moiety.
Archives of Biochemistry and Biophysics, 1998
ping of the whole of exons 2 and 3, whose translation product led to the loss of seven amino acids in the 3-Hydroxy-3-methylglutaric aciduria is a rare recesleader peptide and 57 amino acids in the terminal dosive monogenic disorder that affects ketogenesis and main of the mature enzyme. The association of a nonthe catabolism of L-leucine. We report the biochemical sense mutation with the skipping of the exon that conand molecular characterization of a mutation in the tains it, plus the following exon, is an unusual finding 3-hydroxy-3-methylglutaryl coenzyme A lyase gene in not seen previously in HL deficiencies. The mutation four new probands, three Spanish and one Turkish, described here shows the highest incidence (ú37%) of affected by 3-hydroxy-3-methylglutaric aciduria, all total HL deficiencies reported. ᭧ 1998 Academic Press homozygous for the nonsense mutation Glu37Ter, Key Words: 3-hydroxy 3-methylglutaric aciduria; 3which was reported by our group in two probands of hydroxy 3-methylglutaryl CoA lyase deficiency; ketone Portuguese and Moroccan origin (15). In addition to bodies; leucine metabolism; exonic splicing enhancer; the aberrant mRNAs found in the two previous proexon skipping. bands, a novel species of mature HL mRNA was observed in the patients studied here, since a new cDNA, skipped in exons 2 and 3, was obtained from the mRNAs by reverse-transcription PCR (RT-PCR). Thus, three mRNA species were produced in aberrant splic-3-Hydroxy 3-methylglutaric aciduria is a rare recesings as a result of this nonsense mutation: (i) one of the sive monogenic disorder that affects ketogenesis and expected size that contains the premature stop codon L-leucine metabolism. This disorder appears to be rela-UAA, (ii) another with a deletion of 84 bp correspondtively common in Arabic populations (1, 2). Patients ing to the whole of exon 2, and (iii) a new species found suffering HL deficiency have a diminished capacity to now, with a deletion of 192 bp corresponding to skipsynthesize ketone bodies, which usually produces hypoglycemic states, as a result of fasting or acute illness.
Journal of Inherited Metabolic Disease, 2010
3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients.