The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive

Linda O'Reilly, Peter Bross, Thomas J Corydon, Simon E Olpin, Jakob Hansen, John M Kenney, Shawn E McCandless, Dianne M Frazier, Vibeke Winter, Niels Gregersen, Paul C Engel, Brage Storstein Andresen

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.

OriginalsprogEngelsk
TidsskriftEuropean Journal of Biochemistry
Vol/bind271
Udgave nummer20
Sider (fra-til)4053-63
Antal sider11
ISSN0014-2956
DOI
StatusUdgivet - 2004

Fingeraftryk

Acyl-CoA Dehydrogenase
Screening
Newborn Infant
Mutation
Temperature
Affinity chromatography
Flavoproteins
Proteins
Flavin-Adenine Dinucleotide
Mitochondria
Enzyme Stability
Enzymes
Prosthetics
Purification
Affinity Chromatography
Thermodynamic stability
Coloring Agents
Fatty Acids
Cultured Cells
Oxidation

Citer dette

O'Reilly, Linda ; Bross, Peter ; Corydon, Thomas J ; Olpin, Simon E ; Hansen, Jakob ; Kenney, John M ; McCandless, Shawn E ; Frazier, Dianne M ; Winter, Vibeke ; Gregersen, Niels ; Engel, Paul C ; Andresen, Brage Storstein. / The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive. I: European Journal of Biochemistry. 2004 ; Bind 271, Nr. 20. s. 4053-63.
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title = "The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive",
abstract = "Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.",
keywords = "Acyl-CoA Dehydrogenase, Amino Acid Substitution, Animals, Circular Dichroism, Enzyme Stability, Escherichia coli, Fibroblasts, Glutamic Acid, Histidine, Humans, Infant, Newborn, Lymphocytes, Lysine, Mitochondria, Liver, Models, Molecular, Mutagenesis, Site-Directed, Myristic Acid, Neonatal Screening, Oxidation-Reduction, Rats, Recombinant Proteins, Structure-Activity Relationship, Tyrosine",
author = "Linda O'Reilly and Peter Bross and Corydon, {Thomas J} and Olpin, {Simon E} and Jakob Hansen and Kenney, {John M} and McCandless, {Shawn E} and Frazier, {Dianne M} and Vibeke Winter and Niels Gregersen and Engel, {Paul C} and Andresen, {Brage Storstein}",
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O'Reilly, L, Bross, P, Corydon, TJ, Olpin, SE, Hansen, J, Kenney, JM, McCandless, SE, Frazier, DM, Winter, V, Gregersen, N, Engel, PC & Andresen, BS 2004, 'The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive', European Journal of Biochemistry, bind 271, nr. 20, s. 4053-63. https://doi.org/10.1111/j.1432-1033.2004.04343.x

The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive. / O'Reilly, Linda; Bross, Peter; Corydon, Thomas J; Olpin, Simon E; Hansen, Jakob; Kenney, John M; McCandless, Shawn E; Frazier, Dianne M; Winter, Vibeke; Gregersen, Niels; Engel, Paul C; Andresen, Brage Storstein.

I: European Journal of Biochemistry, Bind 271, Nr. 20, 2004, s. 4053-63.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive

AU - O'Reilly, Linda

AU - Bross, Peter

AU - Corydon, Thomas J

AU - Olpin, Simon E

AU - Hansen, Jakob

AU - Kenney, John M

AU - McCandless, Shawn E

AU - Frazier, Dianne M

AU - Winter, Vibeke

AU - Gregersen, Niels

AU - Engel, Paul C

AU - Andresen, Brage Storstein

PY - 2004

Y1 - 2004

N2 - Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.

AB - Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.

KW - Acyl-CoA Dehydrogenase

KW - Amino Acid Substitution

KW - Animals

KW - Circular Dichroism

KW - Enzyme Stability

KW - Escherichia coli

KW - Fibroblasts

KW - Glutamic Acid

KW - Histidine

KW - Humans

KW - Infant, Newborn

KW - Lymphocytes

KW - Lysine

KW - Mitochondria, Liver

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Myristic Acid

KW - Neonatal Screening

KW - Oxidation-Reduction

KW - Rats

KW - Recombinant Proteins

KW - Structure-Activity Relationship

KW - Tyrosine

U2 - 10.1111/j.1432-1033.2004.04343.x

DO - 10.1111/j.1432-1033.2004.04343.x

M3 - Journal article

VL - 271

SP - 4053

EP - 4063

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

IS - 20

ER -