Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance

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

“…Interestingly, monitoring fluorescence tryptophan emission elicited a similar cooperative behavior and stabilizing effect, although the variation was more discrete (∆T m app = +3 °C, Supplemental Figure S3 ). This smaller change can be explained by the fact that this variant presents looser tertiary structure contacts and, as reported elsewhere [ 23 ], GCDH thermal unfolding probably begins with loss of tertiary structure, which fosters cofactor dissociation followed by secondary structure destabilization, resulting in a CD-monitored higher apparent midpoint unfolding temperature.…”

“…The human GCDH variants are expressed in Escherichia coli using a rich medium (details in material and methods) that is normally sufficient to guarantee that the overexpressed flavoproteins have the FAD cofactor present. Still, as reported previously, during GCDH purification, FAD is routinely added to all the buffers to ensure that purified protein has full occupancy of the cofactor site [ 23 ]. Interestingly, in the case of the GCDH-p.Val400Met variant, during purification of the His-tagged variant by affinity chromatography, the chromatogram showed two peaks between 200 and 300 mM imidazole, corresponding to two distinct bound conformers, in contrast to wild-type GCDH (GCDH-WT) that presented only one bound fraction ( Figure 1 A).…”

“…Previous studies have indicated that the isoalloxazine ring of flavin is a necessary element for the ACDHs to assemble into functional tetramers that would otherwise remain as nonfunctional monomers [ 9 ]. Interestingly, the Val400Met mutation under study is located in an important region that is involved in intersubunit contacts, which implies that lack of flavin combined with the deleterious position of the mutation in the protein structure might cumulatively contribute to influence tetrameric assembly of GCDH [ 23 , 26 ]. Because our experiments indicated that FAD depletion altered protein compactness, we hypothesized that it could have an impact on GCDH quaternary structure.…”

“…The aim of this work was to investigate the molecular mechanism underlying the rescue of GCDH by riboflavin supplementation and subsequently clarify its potential therapeutic effects in GA-I patients. For this, we chose the GCDH-p.Val400Met disease variant as the model, which we have previously reported to present an overall fold similar to that of wild-type, although slightly less compact, with compromised FAD interactions and a substantial residual activity in vitro [ 23 ], making it a good candidate for this investigation. We hypothesized that, as observed for other flavoproteins, improving FAD interactions would have a positive effect on enzyme folding, stability, and function.…”

Deglutarylation of GCDH by SIRT5 controls lysine metabolism in mice

“…In the second assay, the activity of GCDH was inferred from its ability to reduce the electron transfer flavoprotein (ETF), which is its physiological electron acceptor. For this, GCDH was incubated with glutaryl-CoA (15 µM) and purified recombinant human ETF (19,20) and the ability of ETF to receive electrons from GCDH was monitored by measuring DCPIP reduction at 600 nm (21). Buffer for enzymatic assays was 10 mM HEPES pH 7.8.…”