Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients
Abstract
:1. Introduction
2. Results
2.1. Purified Human Recombinant GCDH-p.Val400Met Is Depleted of FAD Cofactor
2.2. Apo GCDH-p.Val400Met Presents a Less Compact Conformation
2.3. Deflavinylation Affects GCDH Quaternary Structure
2.4. Flavin Supplementation Rescues Apo GCDH-p.Val400Met Enzymatic Activity
2.5. Flavin Improves the Conformational Quality of Holo GCDH-p.Val400Met
2.6. External FAD Preserves GCDH-p.Val400Met Enzymatic Activity during Thermal Stress
3. Discussion
4. Materials and Methods
4.1. Chemicals/Materials
4.2. Protein Purification and Biochemical Assays
4.3. Spectroscopic Methods
4.4. Fluorescence Quenching with Acrylamide
4.5. Size-Exclusion Chromatography
4.6. Thermal Stability
4.7. Limited Proteolysis with Trypsin
4.8. Thermal Stress Studies
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
FMN | flavin mononucleotide |
FAD | flavin adenine dinucleotide |
ACDH | acyl-CoA dehydrogenase |
GCDH | glutaryl-CoA dehydrogenase |
GA-I | glutaric aciduria type I |
GABA | γ-aminobutyric acid |
CD | circular dichroism |
AEW | average emission wavelength |
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GCDH | Ka (M−1) | fa |
---|---|---|
Wild-Type | 5.88 ± 0.42 | 0.84 ± 0.04 |
Holo p.Val400Met | 7.43 ± 0.08 | 0.80 ± 0.01 |
Apo p.Val400Met | 8.99 ± 0.33 | 0.89 ± 0.02 |
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Ribeiro, J.V.; Gomes, C.M.; Henriques, B.J. Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients. Int. J. Mol. Sci. 2020, 21, 7063. https://doi.org/10.3390/ijms21197063
Ribeiro JV, Gomes CM, Henriques BJ. Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients. International Journal of Molecular Sciences. 2020; 21(19):7063. https://doi.org/10.3390/ijms21197063
Chicago/Turabian StyleRibeiro, Joana V., Cláudio M. Gomes, and Bárbara J. Henriques. 2020. "Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients" International Journal of Molecular Sciences 21, no. 19: 7063. https://doi.org/10.3390/ijms21197063