Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site

Küchler, Burkhard; Abdel-Ghany, Abdel-Ghany; Bross, Peter; Nandy, Andreas; Rasched, Ihab; Ghisla, Sandro

doi:10.1042/bj3370225

Cited by 15 publications

(11 citation statements)

References 31 publications

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“…The observations obtained from analyzing variants harboring amino acid substitutions in different regions of the protein show that mutations in the β-sheet domain and the adjacent loop (interconnecting the β-domain and the C-terminal α-domain) are particularly instable and prone to severe conformational distortion. This is in line with the previous work describing severely impaired biogenesis and stability for two other variants located in the β-domain, T168A and G170R (c.583G>A) ( 10 , 15 ). Misfolding observed in these variants has been attributed to the loss of FAD anchoring, which has been reported to be crucial for folding and oligomerization ( 34 ).…”

Section: Discussionsupporting

confidence: 93%

“…In addition, the aromatic side chain of Y133 contributes to the hydrophobic core lining the binding cavity for the fatty acid. Previous studies characterizing the molecular phenotype of T168A (c.577A>G), a mutation that has been identified in a clinically diagnosed patient, are in line with our observations revealing misfolding, markedly decreased thermal stability and catalytic activity ( 15 , 16 , 18 ). In contrast, R181C showed a residual catalytic activity comparable to wild-type despite its severe conformational alterations.…”

Section: Discussionsupporting

confidence: 91%

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Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening

Maier

Gersting

Kemter

et al. 2009

Human Molecular Genetics

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Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the β-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 91%

Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening

Maier

Gersting

Kemter

et al. 2009

Human Molecular Genetics

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“…Maxima absorbance ratio (268/444 nm) for IBD found to be ≈ 5.4 (Fig. 1), comparing to 5.7 for human MCAD (Kuechler et al, 1999).…”

Section: Biochemical Characterizationmentioning

confidence: 80%

Biochemical Studies on Recombinant Human Isobutyryl-CoA Dehydrogenase

Ibrahim¹

2011

American Journal of Biochemistry and Biotechnology

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Problem statement: Human Isobutyryl-CoA Dehydrogenase (IBD) is member of the Acyl-CoA Dehydrogenases family. It is involved in Val metabolism. In this study we modified the purification method of the IBD and further spectroscopic characterization was conducted. Approach: Using DTT in the buffers during the purification, IBD purified to homogeneity by different chromatographic steps. Reactivity of Cys residues were measured using thiol modifying agent DNTB. Results: IBD found to be homotetramer with 42.7 KDa for each subunit and the value of its pI was 6.2. Conclusion: Thiol modifying reagent showed crucial role (s) of some Cys residues for IBD structure, FAD binding and/or activity

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“…The activity, determined experimentally by a ferricinium assay of this mutant enzyme, was reduced by only 80% of that of the wild-type (11). This translates to an increase in the phenomenological free energy of activation by less than 1 kcal/mol.…”

Section: 22mentioning

confidence: 81%

Potential of Mean Force Calculation for the Proton and Hydride Transfer Reactions Catalyzed by Medium-Chain Acyl-CoA Dehydrogenase: Effect of Mutations on Enzyme Catalysis

Bhattacharyya

Stankovich

et al. 2005

Biochemistry

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Potential of mean force calculations have been performed on the wild-type medium chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. Initial simulation and analysis of the active site of the enzyme reveals that an arginine residue (Arg256), conserved in the substrate binding domain of this group of enzymes, exists in two alternate conformations, only one of which makes the enzyme active. This active conformation was used in subsequent computations of the enzymatic reactions. It is known that the catalytic α,β-dehydrogenation of fatty acyl-CoAs consists of two C-H bond dissociation processes: a proton abstraction and a hydride transfer. Energy profiles of the two reaction steps in the wild-type MCAD demonstrate that the reaction proceeds by a stepwise mechanism with a transient species. The activation barriers of the two steps differ by only ∼2 kcal/ mol, indicating that both may contribute to the rate-limiting process. Thus this may be a stepwise dissociation mechanism whose relative barriers can be tuned by suitable alterations of the substrate and/or enzyme. Analysis of the structures along the reaction path reveals that Arg256 plays a key role in maintaining the reaction-center hydrogen-bonding network involving the thioester carbonyl group, which stabilizes transition states as well as the intervening transient species. Mutation of this arginine residue to glutamine increases the activation barrier of the hydride transfer reaction by ∼5 kcal/mol, and the present simulations predict a substantial loss of catalytic activity for this mutant. Structural analysis of this mutant reveals that the orientation of the thioester moiety of the substrate has been changed significantly as compared to that in the wild-type enzyme. In contrast, simulation of the active site of the Thr168Ala mutant shows no significant change in the relative orientation of the substrate and the cofactor in the active site; as a result, this mutation has very little effect on the overall reaction barrier, and this is consistent with the experimental data. This study demonstrates that significant insights of the catalytic mechanism can be obtained by these simulated enzyme catalysis studies whose results can pave the way of designing novel mechanistic probes for the enzyme.Mitochondrial β-oxidation has been extensively studied in the past 15 years because a number of mutations in several of the enzymes involved are responsible for diseases related to fatty acid metabolism. Fatty acid metabolism occurs within the mitochondrial matrix and proceeds by a sequence of steps that remove two carbon units in each cycle. The process involves at a This work was partially supported by grant GM 29344 from the National Institutes of Healths to MTS, by grant GM046736 from National Institutes of Healths to JG, by NSF grant CHE03−49122 to DGT. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript least 12 different enzymes, including the acyl-CoA dehydrogenases, which are flavoenzymes that perform the first step of thi...

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Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site

Cited by 15 publications

References 31 publications

Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening

Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening

Biochemical Studies on Recombinant Human Isobutyryl-CoA Dehydrogenase

Potential of Mean Force Calculation for the Proton and Hydride Transfer Reactions Catalyzed by Medium-Chain Acyl-CoA Dehydrogenase: Effect of Mutations on Enzyme Catalysis

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