Affinage

ACAD9

Complex I assembly factor ACAD9, mitochondrial · UniProt Q9H845

Length
621 aa
Mass
68.8 kDa
Annotated
2026-06-09
26 papers in source corpus 10 papers cited in narrative 11 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/6 claims corpus-supported (83%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ACAD9 is a mitochondrial flavoenzyme with dual, mutually exclusive functions: as an acyl-CoA dehydrogenase in long-chain fatty acid β-oxidation and as an essential assembly factor for respiratory chain Complex I (PMID:12359260, PMID:21057504, PMID:34646991). As an enzyme, it catalyzes α,β-dehydrogenation of long-chain acyl-CoA esters, with maximal activity toward unsaturated long-chain substrates, and contributes physiologically to fatty acid oxidation in high-expressing tissues such as liver and neurons; this activity is non-redundant with VLCAD and generates specific acylcarnitine species (C14:1 from oleate, C12 from palmitate) (PMID:12359260, PMID:17564966, PMID:24158852, PMID:25721401). Independently of its catalytic activity, ACAD9 functions as a Complex I assembly chaperone, since catalytically inactive ACAD9 still rescues Complex I biogenesis and is incorporated into high-molecular-weight assembly intermediates (PMID:24158852). ACAD9 executes this role by forming a stable, soluble ternary complex with ECSIT and NDUFAF1, binding the C-terminal half of ECSIT while NDUFAF1 binds its N-terminal half; ECSIT binds at the ETF-binding site of ACAD9, displacing FAD and abolishing dehydrogenase activity, which explains the mutual exclusivity of the two functions (PMID:34646991). ACAD9 also stabilizes ECSIT protein in vivo (PMID:34556413). Pathogenic ACAD9 mutations cause isolated Complex I deficiency that is corrected by wild-type but not mutant ACAD9, with clinical severity correlating inversely with residual enzymatic activity (PMID:21057504, PMID:20929961, PMID:25721401).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 2002 Medium

    Established ACAD9 as a bona fide acyl-CoA dehydrogenase, defining its molecular activity before any role in disease was known.

    Evidence Cloning and in vitro enzymatic assay of recombinant ACAD9 on palmitoyl-CoA and stearoyl-CoA

    PMID:12359260

    Open questions at the time
    • Single in vitro assay does not establish in vivo substrate preference
    • No structural or cofactor characterization
    • No link to physiology or disease
  2. 2007 Medium

    Showed ACAD9 has distinct, non-redundant function in long-chain fat metabolism, ruling out simple compensation by VLCAD despite overlapping substrate range.

    Evidence Biochemical substrate specificity assays plus patient fibroblast/tissue acylcarnitine profiling

    PMID:17564966

    Open questions at the time
    • Mechanistic basis of non-redundancy unresolved at this stage
    • No molecular partner identified
    • Single-lab patient series
  3. 2010 High

    Reassigned ACAD9 as an essential Complex I assembly factor, demonstrating a function beyond fatty acid oxidation through restoration of Complex I in patient cells.

    Evidence Exome sequencing/homozygosity mapping with wild-type vs mutant complementation in patient fibroblasts; riboflavin response

    PMID:20929961 PMID:21057504

    Open questions at the time
    • Did not separate enzymatic from assembly contributions
    • Molecular partners in assembly unknown
    • Mechanism of Complex I rescue undefined
  4. 2013 High

    Resolved whether catalysis is needed for the chaperone role, showing the two functions are separable, and pinpointed which acylcarnitine species ACAD9 generates.

    Evidence Catalytically inactive ACAD9 complementation with BN-PAGE assembly-intermediate analysis; knockdown in VLCAD-deficient fibroblasts with acylcarnitine profiling

    PMID:24158852

    Open questions at the time
    • Identity of assembly partners still not defined
    • Structural basis of incorporation into intermediates unknown
  5. 2015 High

    Demonstrated ACAD9 contributes physiologically to long-chain fatty acid oxidation and that residual enzymatic activity of mutations predicts clinical severity, unifying the enzyme and disease readouts.

    Evidence ACAD9 knockout in HEK293 cells with FAO flux and Complex I assays; prokaryotic enzymatic assay of 16 pathogenic mutations correlated with patient severity

    PMID:25721401

    Open questions at the time
    • Tissue-level FAO contribution in vivo not directly measured
    • Correlation does not establish causal mechanism per mutation
  6. 2021 High

    Provided the structural and biochemical basis for mutual exclusivity, defining the ACAD9-ECSIT-NDUFAF1 ternary assembly complex and how ECSIT binding displaces FAD.

    Evidence Reconstitution of binary/ternary complexes, SAXS and modelling, FAD release and enzymatic assays, mapping of 42 mutations

    PMID:34646991

    Open questions at the time
    • High-resolution structure of the ternary complex not determined
    • Dynamics of switching between FAO and assembly states in cells unresolved
  7. 2021 High

    Established the in vivo physiological requirement and tissue-specificity of ACAD9 and confirmed its role in stabilizing ECSIT.

    Evidence Cardiac- and muscle-specific Cre-lox knockout mice with phenotyping, mitochondrial assays, and ECSIT Western blot

    PMID:34556413

    Open questions at the time
    • Mechanism of tissue-specific severity differences unexplained
    • Relative contribution of FAO vs assembly loss to cardiac phenotype not dissected
  8. 2022 Medium

    Identified a variant (V546M) that impairs Complex I activity without altering complex amount, indicating ACAD9 can affect Complex I function beyond assembly/stability.

    Evidence Expression of V546M variant in cells with respiration, ATP, and BN-PAGE/SDS-PAGE assays

    PMID:40806260

    Open questions at the time
    • Single variant in single lab
    • Mechanism by which activity is impaired without assembly defect unknown
  9. 2025 Medium

    Extended ACAD9 function to redox and lipid homeostasis, showing its loss redirects linoleic acid flux and promotes ferroptosis in cancer cells.

    Evidence In vivo genome-wide CRISPR screen in orthotopic ovarian cancer model with multi-omics and KO mechanistic studies

    PMID:40618880

    Open questions at the time
    • Single-lab/single-cancer-context study
    • Direct vs indirect role in linoleic acid handling not separated from Complex I collapse

Open questions

Synthesis pass · forward-looking unresolved questions
  • How cells regulate the switch between ACAD9's enzymatic and assembly states, and a high-resolution structure of the ternary assembly complex, remain open.
  • No high-resolution ternary complex structure
  • Regulatory determinants of FAO-vs-assembly partitioning in vivo unknown
  • Mechanism of variant-specific Complex I activity loss undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016491 oxidoreductase activity 3 GO:0044183 protein folding chaperone 2 GO:0016740 transferase activity 1
Localization
GO:0005739 mitochondrion 3
Pathway
R-HSA-1430728 Metabolism 3 R-HSA-1852241 Organelle biogenesis and maintenance 3
Partners
ECSITNDUFAF1
Complex memberships
ACAD9-ECSIT-NDUFAF1 complex I assembly complex

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2002 ACAD9 encodes a novel acyl-CoA dehydrogenase (ninth member of the ACAD family) with dehydrogenase enzymatic activity on palmitoyl-CoA (C16:0) and stearoyl-CoA (C18:0), confirmed by enzymatic assay of recombinant protein. Enzymatic assay of recombinant ACAD9 protein; cloning and sequence analysis identifying ACAD family signatures Biochemical and biophysical research communications Medium 12359260
2007 ACAD9 demonstrates maximum catalytic activity with unsaturated long-chain acyl-CoAs; despite overlapping substrate specificity with VLCAD, ACAD9 and VLCAD cannot compensate for each other in vivo, indicating two independently regulated functional pathways for long-chain fat metabolism. Biochemical substrate specificity assays; patient fibroblast/tissue studies showing distinct acylcarnitine profiles; ACAD9 mRNA/protein defect characterization in patients American journal of human genetics Medium 17564966
2010 ACAD9 is required for mitochondrial respiratory chain complex I assembly; pathogenic ACAD9 mutations cause isolated complex I deficiency, and re-expression of wild-type ACAD9 in patient-derived fibroblasts corrects the complex I defect. Whole-exome sequencing to identify mutations; complementation of complex I defect by wild-type ACAD9 expression in patient fibroblasts Nature genetics High 21057504
2010 A homozygous ACAD9 mutation (R532W) causes complex I deficiency; wild-type but not mutant ACAD9 restores complex I activity when transduced into patient fibroblasts, confirming the essential role of ACAD9 in complex I function. Riboflavin supplementation improved complex I activity. Homozygosity mapping; lentiviral transduction complementation assay in patient fibroblasts with wild-type vs. mutant ACAD9; protein modelling Brain : a journal of neurology High 20929961
2013 Catalytically inactive ACAD9 provides partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and is incorporated into high-molecular-weight complex I assembly intermediates, demonstrating that ACAD9 enzymatic activity is not required for its complex I assembly chaperone function. Knockdown/complementation in ACAD9-deficient fibroblasts using catalytically inactive ACAD9 mutant; BN-PAGE analysis of assembly intermediates; acylcarnitine profiling Human molecular genetics High 24158852
2013 ACAD9 knockdown in VLCAD-deficient fibroblasts revealed that ACAD9 is responsible for production of C14:1-carnitine from oleate and C12-carnitine from palmitate, explaining obscure acylcarnitine species used to diagnose VLCAD deficiency. Stable knockdown in VLCAD-deficient fibroblasts; acylcarnitine profiling upon fatty acid loading Human molecular genetics Medium 24158852
2015 ACAD9 plays a physiological role in long-chain fatty acid oxidation in cells expressing high ACAD9 levels (e.g., HEK293 cells, liver, neurons); ACAD9 knockout in HEK293 cells impaired both long-chain fatty acid oxidation and complex I activity, both rescued by wild-type ACAD9. Residual ACAD enzymatic activity of patient mutations inversely correlates with clinical severity. ACAD9 knockout in HEK293 cells; fatty acid oxidation flux assays; complementation with wild-type ACAD9; prokaryotic expression system to measure ACAD activity of 16 pathogenic mutations; correlation analysis with patient phenotype severity Human molecular genetics High 25721401
2021 ACAD9 forms a ternary complex with ECSIT and NDUFAF1 as the core mitochondrial complex I assembly complex. ACAD9 binds the carboxy-terminal half of ECSIT, while NDUFAF1 binds the amino-terminal half. Binary ACAD9/ECSIT or NDUFAF1/ECSIT complexes are unstable and aggregate, whereas the ternary complex is soluble and highly stable. ECSIT binding occurs at the ETF binding site in the amino-terminal domain of ACAD9, resulting in loss of FAD and enzymatic activity, demonstrating that ACAD9's two functions (FAO and complex I assembly) are mutually exclusive. Protein binding studies (binary and ternary complex formation); small-angle X-ray scattering (SAXS); molecular modelling; FAD release and enzymatic activity assays; mapping of 42 pathogenic mutations onto homology model iScience High 34646991
2021 Cardiac-specific ACAD9 knockout mice develop severe neonatal cardiomyopathy and die by 17 days; ECSIT protein levels are significantly reduced in the absence of ACAD9, confirming that ACAD9 is required to stabilize ECSIT in the complex I assembly pathway. Muscle-specific ACAD9 knockout mice are viable but exhibit muscle weakness. Cre-lox tissue-specific knockout mouse models; Western blot for ECSIT; in vitro mitochondrial function assays; histological analysis Molecular genetics and metabolism High 34556413
2025 ACAD9 preserves electron transport chain (complex I) integrity and regulates linoleic acid metabolism for energy production and ROS mitigation in ovarian cancer cells; ACAD9 loss triggers mitochondrial respiratory collapse, ROS accumulation, and redirects linoleic acid flux from β-oxidation toward membrane lipid biosynthesis, increasing polyunsaturated fatty acid incorporation and promoting ferroptosis. In vivo CRISPR/Cas9 genome-wide knockout screen in orthotopic mouse model; multi-omics (metabolomics, lipidomics, transcriptomics); ACAD9 KO mechanistic studies in cancer cell lines Cancer letters Medium 40618880
2022 Expression of ACAD9 V546M variant in cell lines reduces mitochondrial complex I activity by over 50% without affecting the total amount of respiratory chain complexes, indicating this variant specifically impairs complex I activity rather than complex assembly or stability. Molecular cloning and expression of ACAD9 V546M variant in cell line; mitochondrial respiration assays; ATP production measurement; BN-PAGE/SDS-PAGE for OXPHOS complex composition International journal of molecular sciences Medium 40806260

Source papers

Stage 0 corpus · 26 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2010 Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency. Nature genetics 187 21057504
2007 A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency. American journal of human genetics 98 17564966
2010 Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective ACAD9: new function for an old gene. Brain : a journal of neurology 93 20929961
2002 Cloning and functional characterization of ACAD-9, a novel member of human acyl-CoA dehydrogenase family. Biochemical and biophysical research communications 75 12359260
2018 Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective? Orphanet journal of rare diseases 57 30025539
2013 ACAD9, a complex I assembly factor with a moonlighting function in fatty acid oxidation deficiencies. Human molecular genetics 57 24158852
2015 Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency. Human molecular genetics 45 25721401
2013 Mitochondrial encephalomyopathy due to a novel mutation in ACAD9. JAMA neurology 40 23836383
2013 A Patient with Complex I Deficiency Caused by a Novel ACAD9 Mutation Not Responding to Riboflavin Treatment. JIMD reports 28 23996478
2015 Neonatal multiorgan failure due to ACAD9 mutation and complex I deficiency with mitochondrial hyperplasia in liver, cardiac myocytes, skeletal muscle, and renal tubules. Human pathology 27 26826406
2015 High incidence and variable clinical outcome of cardiac hypertrophy due to ACAD9 mutations in childhood. European journal of human genetics : EJHG 25 26669660
2021 Molecular mechanism of interactions between ACAD9 and binding partners in mitochondrial respiratory complex I assembly. iScience 24 34646991
2016 Evidence of a wide spectrum of cardiac involvement due to ACAD9 mutations: Report on nine patients. Molecular genetics and metabolism 23 27233227
2016 An atypical presentation of ACAD9 deficiency: Diagnosis by whole exome sequencing broadens the phenotypic spectrum and alters treatment approach. Molecular genetics and metabolism reports 12 28070495
2021 Development and characterization of a mouse model for Acad9 deficiency. Molecular genetics and metabolism 11 34556413
2025 Metabolic gatekeeper ACAD9 coordinates linoleic acid metabolism and redox homeostasis via mitochondrial complex I to drive ovarian cancer progression. Cancer letters 9 40618880
2023 Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A). Frontiers in immunology 9 37388727
2022 Molecular characteristics of the multi-functional FAO enzyme ACAD9 illustrate the importance of FADH2 /NADH ratios for mitochondrial ROS formation. BioEssays : news and reviews in molecular, cellular and developmental biology 9 35708204
2017 Severe defect in mitochondrial complex I assembly with mitochondrial DNA deletions in ACAD9-deficient mild myopathy. Muscle & nerve 9 27438479
2017 Lifetime exercise intolerance with lactic acidosis as key manifestation of novel compound heterozygous ACAD9 mutations causing complex I deficiency. Neuromuscular disorders : NMD 9 28279569
2017 Assembly defects of multiple respiratory chain complexes in a child with cardiac hypertrophy associated with a novel ACAD9 mutation. Molecular genetics and metabolism 9 28529009
2015 Lethal Neonatal Progression of Fetal Cardiomegaly Associated to ACAD9 Deficiency. JIMD reports 9 26475292
2022 Severe Antenatal Hypertrophic Cardiomyopathy Secondary to ACAD9-Related Mitochondrial Complex I Deficiency. Molecular syndromology 7 37064341
2010 Assembling complex I with ACAD9. Cell metabolism 4 20816087
2020 Successful pregnancy in a patient with mitochondrial cardiomyopathy due to ACAD9 deficiency. JIMD reports 3 33204590
2025 A Late-Onset and Mild Phenotype of Mitochondrial Complex I Deficiency Due to a Novel Reported Variant Within the ACAD9 Gene. International journal of molecular sciences 2 40806260

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