Journal of Inherited Metabolic Disease

, Volume 37, Issue 6, pp 917–928 | Cite as

Functional studies of 18 heterologously expressed medium-chain acyl-CoA dehydrogenase (MCAD) variants

  • Kira-Lee Koster
  • Marga Sturm
  • Diran Herebian
  • Sander H. J. Smits
  • Ute Spiekerkoetter
Original Article


Medium-chain acyl-coenzyme-A dehydrogenase (MCAD) catalyzes the first step of mitochondrial beta-oxidation for medium-chain acyl-CoAs. Mutations in the ACADM gene cause MCAD deficiency presenting with life-threatening symptoms during catabolism. Since fatty-acid-oxidation disorders are part of newborn screening (NBS), many novel mutations with unknown clinical relevance have been identified in asymptomatic newborns. Eighteen of these mutations were separately cloned into the human ACADM gene, heterologously overexpressed in Escherichia coli and functionally characterized by using different substrates, molecular chaperones, and measured at different temperatures. In addition, they were mapped to the three-dimensional MCAD structure, and cross-link experiments were performed. This study identified variants that only moderately affect the MCAD protein in vitro, such as Y42H, E18K, and R6H, in contrast to the remaining 15 mutants. These three mutants display residual octanoyl-CoA oxidation activities in the range of 22 % to 47 %, are as temperature sensitive as the wild type, and reach 100 % activity with molecular chaperone co-overexpression. Projection into the three-dimensional protein structure gave some indication as to possible reasons for decreased enzyme activities. Additionally, six of the eight novel mutations, functionally characterized for the first time, showed severely reduced residual activities < 5 % despite high expression levels. These studies are of relevance because they classify novel mutants in vitro on the basis of their corresponding functional effects. This basic knowledge should be taken into consideration for individual management after NBS.


Residual Activity Molecular Chaperone Flavin Adenine Dinucleotide PyMOL Molecular Graphic System Neighboring Monomer 



Medium-chain acyl-CoA dehydrogenase deficiency


Newborn screening


Flavin adenine dinucleotide


Coenzyme A


Bis(sulfosuccinimidyl) suberate



We kindly acknowledge Annette Seibt for technical advice and support.

Conflict of interest


Supplementary material

10545_2014_9732_MOESM1_ESM.doc (50 kb)
Supplemental table S1 (DOC 50 kb)
10545_2014_9732_Fig6_ESM.gif (114 kb)
Supplemental ESM2

Oligomerization of wild-type and mutant MCAD: Extracts of wild type and mutant MCAD proteins after cross-linkage with (+) and without (-) cross-linker BS³, SDS-PAGE and western blot analysis. The positions of monomer (67 kDa), dimers (134 kDa) and tetramers (268 kDa) are indicated. The identities of the heteromeric MCAD-immunoreactive complexes are unknown. Three independent experiments were performed (GIF 113 kb)

10545_2014_9732_MOESM2_ESM.tif (176 kb)
High Resolution (TIFF 176 kb)


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Copyright information

© SSIEM 2014

Authors and Affiliations

  • Kira-Lee Koster
    • 1
  • Marga Sturm
    • 1
  • Diran Herebian
    • 1
  • Sander H. J. Smits
    • 2
  • Ute Spiekerkoetter
    • 3
  1. 1.Department of General Pediatrics, Neonatology and Pediatric CardiologyUniversity Children’s HospitalDuesseldorfGermany
  2. 2.Institute of BiochemistryHeinrich-Heine-UniversityDuesseldorfGermany
  3. 3.Department of General Pediatrics, Center for Pediatrics and Adolescent MedicineUniversity HospitalFreiburgGermany

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