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

Riboflavin is the biological precursor of two important flavin cofactors—flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)—that are critical prosthetic groups in several redox enzymes. While dietary supplementation with riboflavin is a recognized support therapy in several inborn errors of metabolism, it has yet unproven benefits in several other pathologies affecting flavoproteins. This is the case for glutaric aciduria type I (GA-I), a rare neurometabolic disorder associated with mutations in the GCDH gene, which encodes for glutaryl-coenzyme A (CoA) dehydrogenase (GCDH). Although there are a few reported clinical cases that have responded to riboflavin intake, there is still not enough molecular evidence supporting therapeutic recommendation. Hence, it is necessary to elucidate the molecular basis in favor of riboflavin supplementation in GA-I patients. Here, using a combination of biochemical and biophysical methodologies, we investigate the clinical variant GCDH-p.Val400Met as a model for a phenotype associated with severe deflavinylation. Through a systematic analysis, we establish that recombinant human GCDH-p.Val400Met is expressed in a nonfunctional apo form, which is mainly monomeric rather than tetrameric. However, we show that exogenous FAD is a driver for structural reorganization of the mutant enzyme with concomitant functional recovery, improved thermolability, and resistance to trypsin digestion. Overall, these results establish proof of principle for the beneficial effects of riboflavin supplementation in GA-I patients.

Glutaric aciduria and L-2-hydroxyglutaric aciduria: Clinical and molecular findings of 35 patients from Turkey

“…Also, the P248L variant is associated with some residual GCDH activity in other patients. In addition, in the p.Val400Met homozygous mutation study, riboflavin was reported to be a driving force for the mutant enzyme [ 23 ]. These results provide principal proof for the beneficial effects of riboflavin in GA-I.…”