Journal Article

PUBDB-2025-04615

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png High-resolution native electrophoresis in-gel activity assay reveals biological insights of medium-chain fatty acyl-CoA dehydrogenase deficiency

Guerrero-Castillo, S. (Corresponding author)Extern* ; Grün, A. ; Lewandowski, N. ; Gundorova, P. ; Blettenberger, L. E. ; Laubach, N. C. ; Küchler, K. ; Barroso, M. ; Uetrecht, C.CSSB*XFEL.EU* ; Gersting, S. W. (Corresponding author)

2025
Springer Nature [London]

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Please use a persistent id in citations: doi:10.1038/s41598-025-24684-3  doi:10.3204/PUBDB-2025-04615

Abstract: Medium-chain specific acyl-CoA dehydrogenase (MCAD) is a mitochondrial homotetrameric flavoprotein that catalyzes the first step in fatty acid beta-oxidation. MCAD deficiency arises from variants that either impair enzymatic activity or destabilize interactions between subunits, leading to protein aggregation. Standard enzymatic assays measure the overall MCAD activity but cannot differentiate between tetramers and other protein forms—critical for understanding the impact of pathogenic variants on structure destabilization. In this study, we adapted a native gel colorimetric assay to quantify the activity of MCAD tetramers separately from other protein forms, providing novel insights into how pathogenic variants affect MCAD structure and function. The assay showed a linear correlation between protein amount and enzymatic activity for octanoyl-CoA, a physiological MCAD substrate. Applying this method to clinically relevant MCAD variants allowed us to distinguish subtle differences in protein shape, enzymatic activity, and FAD content, offering profound implications for understanding the molecular basis of MCADD. This methodology can be extended to analyze variants in other acyl-CoA dehydrogenase family members—such as glutaryl-CoA, isovaleryl-CoA or short-chain fatty acyl-CoA dehydrogenases—that are implicated in disorders of fatty acid and amino acid metabolism.

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Contributing Institute(s):

1. CSSB - Leibniz-Institut für Experimentelle Virologie (LIV) / DESY - Charlotte Uetrecht (CSSB-LIV/DESY-CU)

Research Program(s):

1. 633 - Life Sciences – Building Blocks of Life: Structure and Function (POF4-633) (POF4-633)
2. SPOCkS MS - Sampling Protein cOmplex Conformational Space with native top down Mass Spectrometry (759661) (759661)

Experiment(s):

1. No specific instrument

Appears in the scientific report 2025

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