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

Glutaric Aciduria Type I (GA-I), is an autosomal recessive neurometabolic disease caused by mutations in the GCDH gene that encodes for glutaryl-CoA dehydrogenase (GCDH), a flavoprotein involved in the metabolism of tryptophan, lysine and hydroxylysine. Although over 200 disease mutations have been reported a clear correlation between genotype and phenotype has been difficult to establish. To contribute to a better molecular understanding of GA-I we undertook a detailed molecular study on two GCDH disease-related variants, GCDH-p.Arg227Pro and GCDH-p.Val400Met. Heterozygous patients harbouring these two mutations have increased residual enzymatic activity in relation to homozygous patients with only one of the mutations, suggesting a complementation effect between the two. Combining biochemical, biophysical and structural methods we here establish the effects of these mutations on protein folding, stability and catalytic activity. We show that both variants retain the overall protein fold, but with compromised enzymatic activities. Detailed enzyme kinetic studies reveal that GCDH-p.Arg227Pro has impaired function due to deficient substrate affinity as evidenced by its higher K

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