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

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Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance. / Ribeiro, Joana V; Lucas, Tânia G; Bross, Peter; Gomes, Cláudio M; Henriques, Bárbara J.

I: B B A - Proteins and Proteomics, Bind 1868, Nr. 1, 140269, 2020.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{ae2682ee9238404da84b9e1a2e49b6ab,
title = "Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance",
abstract = "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 Km, and that the lower activity in GCDH-p.Val400Met results from weaker interactions with its physiological redox partner (electron transfer flavoprotein). Moreover, the GCDH-p.Val400Met variant has a significantly lower thermal stability (ΔTm ≈ 9 °C), and impaired binding of the FAD cofactor in relation to wild-type protein. On these grounds, we provide a rational for the possible interallelic complementation observed in heterozygous patients based on the fact that in GCDH, the low active p.Arg227Pro variant contributes to stabilize the tetramer while the structurally unstable p.Val400Met variant compensates for enzyme activity.",
keywords = "Flavoprotein, Glutaric aciduria type I, Glutaryl-CoA dehydrogenase, Neurometabolic disorder, Protein folding, Spectroscopy",
author = "Ribeiro, {Joana V} and Lucas, {T{\^a}nia G} and Peter Bross and Gomes, {Cl{\'a}udio M} and Henriques, {B{\'a}rbara J}",
note = "Copyright {\textcopyright} 2019 Elsevier B.V. All rights reserved.",
year = "2020",
doi = "10.1016/j.bbapap.2019.140269",
language = "English",
volume = "1868",
journal = "B B A - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier BV",
number = "1",

}

RIS

TY - JOUR

T1 - Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance

AU - Ribeiro, Joana V

AU - Lucas, Tânia G

AU - Bross, Peter

AU - Gomes, Cláudio M

AU - Henriques, Bárbara J

N1 - Copyright © 2019 Elsevier B.V. All rights reserved.

PY - 2020

Y1 - 2020

N2 - 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 Km, and that the lower activity in GCDH-p.Val400Met results from weaker interactions with its physiological redox partner (electron transfer flavoprotein). Moreover, the GCDH-p.Val400Met variant has a significantly lower thermal stability (ΔTm ≈ 9 °C), and impaired binding of the FAD cofactor in relation to wild-type protein. On these grounds, we provide a rational for the possible interallelic complementation observed in heterozygous patients based on the fact that in GCDH, the low active p.Arg227Pro variant contributes to stabilize the tetramer while the structurally unstable p.Val400Met variant compensates for enzyme activity.

AB - 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 Km, and that the lower activity in GCDH-p.Val400Met results from weaker interactions with its physiological redox partner (electron transfer flavoprotein). Moreover, the GCDH-p.Val400Met variant has a significantly lower thermal stability (ΔTm ≈ 9 °C), and impaired binding of the FAD cofactor in relation to wild-type protein. On these grounds, we provide a rational for the possible interallelic complementation observed in heterozygous patients based on the fact that in GCDH, the low active p.Arg227Pro variant contributes to stabilize the tetramer while the structurally unstable p.Val400Met variant compensates for enzyme activity.

KW - Flavoprotein

KW - Glutaric aciduria type I

KW - Glutaryl-CoA dehydrogenase

KW - Neurometabolic disorder

KW - Protein folding

KW - Spectroscopy

UR - http://www.scopus.com/inward/record.url?scp=85072215716&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2019.140269

DO - 10.1016/j.bbapap.2019.140269

M3 - Journal article

C2 - 31491587

VL - 1868

JO - B B A - Proteins and Proteomics

JF - B B A - Proteins and Proteomics

SN - 1570-9639

IS - 1

M1 - 140269

ER -