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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Published on: November 22, 2014
Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis: substrate specificity and coenzyme A binding
François Talfournier1, Claire Stines-Chaumeil, Guy Branlant
1Unité Mixte de Recherche CNRS, Université Henri Poincaré 7214 AREMS, ARN-RNP Structure-Fonction-Maturation, Enzymologie Moléculaire et Structurale, Nancy Université, Faculté des Sciences et Technologies, Bd. des Aiguillettes, BP 70239, 54506 Vandœuvre-lès-Nancy Cedex, France. francois.talfournier@maem.uhp-nancy.fr
The Journal of Biological Chemistry
|April 26, 2011
Summary
Methylmalonate-semialdehyde dehydrogenase (MSDH) uses specific arginines for substrate binding and enhances CoA reactivity. This study clarifies MSDH
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein-ligand interactions
Background:
- Methylmalonate-semialdehyde dehydrogenase (MSDH) is a unique enzyme in the aldehyde dehydrogenase superfamily.
- MSDH catalyzes the conversion of methylmalonate semialdehyde (MMSA) to propionyl-CoA, involving β-decarboxylation.
- Previous work identified β-decarboxylation as rate-limiting, occurring before CoA attack.
Purpose of the Study:
- To elucidate the molecular basis of MMSA carboxylate recognition by MSDH.
- To investigate how CoA binding influences MSDH's reactivity.
- To determine kinetic parameters for transthioesterification.
Main Methods:
- Site-directed mutagenesis of invariant arginines (Arg-124, Arg-301) to Leucine.
- Kinetic analysis of enzyme acylation and deacylation steps.
- Substrate substitution using propionaldehyde to study transthioesterification.
Main Results:
- Mutating Arg-124 and Arg-301 significantly reduced MMSA binding and acylation efficiency (by >50-fold).
- CoA binding to MSDH lowers its apparent pK(app) by ~3 units during deacylation.
- Transthioesterification catalytic efficiency increased by >10,000-fold compared to a chemical model.
- CoA binding was observed with the acylation complex, suggesting a distinct binding site from NAD(H).
Conclusions:
- Invariant arginines are crucial for MMSA recognition and binding.
- MSDH significantly enhances CoA reactivity and catalytic efficiency through specific binding interactions.
- A distinct CoA binding site exists, separate from the NAD(H) site, influencing enzyme mechanism.
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