Affinage

ACAD9

Complex I assembly factor ACAD9, mitochondrial · UniProt Q9H845

Length
621 aa
Mass
68.8 kDa
Annotated
2026-04-28
26 papers in source corpus 11 papers cited in narrative 12 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ACAD9 is a dual-function mitochondrial flavoenzyme that participates in both long-chain fatty acid β-oxidation and mitochondrial respiratory chain complex I assembly. As an acyl-CoA dehydrogenase, ACAD9 catalyzes α,β-dehydrogenation of long-chain (C16–C18) and preferentially unsaturated fatty acyl-CoAs in a pathway non-redundant with VLCAD (PMID:12359260, PMID:17564966, PMID:24158852). Independently of its enzymatic activity, ACAD9 functions as an essential complex I assembly factor by forming a stable ternary complex with ECSIT and NDUFAF1, where ECSIT binding at the ETF-interaction site displaces FAD and abolishes dehydrogenase activity, rendering the two functions mutually exclusive; loss of ACAD9 destabilizes ECSIT and causes severe complex I deficiency (PMID:34646991, PMID:24158852, PMID:34556413). Biallelic loss-of-function mutations in ACAD9 cause mitochondrial complex I deficiency presenting as hypertrophic cardiomyopathy and exercise intolerance, with residual enzymatic activity inversely correlating with clinical severity (PMID:21057504, PMID:20929961, PMID:25721401).

Mechanistic history

Synthesis pass · year-by-year structured walk · 7 steps
  1. 2002 High

    Establishing ACAD9 as a bona fide mitochondrial acyl-CoA dehydrogenase resolved the molecular identity of the gene product: recombinant ACAD9 catalyzed dehydrogenation of palmitoyl-CoA and stearoyl-CoA, placing it within the long-chain acyl-CoA dehydrogenase family.

    Evidence In vitro enzymatic assay with recombinant human ACAD9 on C16:0 and C18:0 substrates

    PMID:12359260

    Open questions at the time
    • Substrate preference for unsaturated vs. saturated acyl-CoAs not yet defined
    • Physiological redundancy with VLCAD unknown
    • No structural data available
  2. 2007 Medium

    Demonstrating that ACAD9 and VLCAD do not compensate for each other in patient cells established that ACAD9 operates in a distinct fatty acid oxidation pathway, preferring unsaturated long-chain substrates.

    Evidence Biochemical and substrate specificity analyses in patient-derived cells deficient in either ACAD9 or VLCAD

    PMID:17564966

    Open questions at the time
    • No genetic complementation to confirm causality
    • Contribution of ACAD9 to total cellular long-chain FAO not quantified
  3. 2010 High

    The unexpected discovery that ACAD9 is required for mitochondrial complex I assembly — demonstrated by complementation rescue in patient fibroblasts — revealed a second, non-enzymatic function and explained why ACAD9 mutations cause complex I deficiency and cardiomyopathy.

    Evidence Lentiviral expression of wild-type vs. mutant (R532W) ACAD9 in patient fibroblasts; complex I activity measurement; multiple patient lines across two independent studies

    PMID:20929961 PMID:21057504

    Open questions at the time
    • Mechanism of complex I assembly role unknown
    • Identity of binding partners in assembly pathway not defined
    • Whether enzymatic and assembly functions are separable not established
  4. 2013 High

    Separation-of-function experiments resolved the dual-function question: catalytically dead ACAD9 rescued complex I assembly, while knockdown in VLCAD-deficient cells confirmed ACAD9's non-redundant contribution to long-chain fatty acid oxidation, proving the two roles are mechanistically independent.

    Evidence Active-site mutagenesis with BN-PAGE and complex I assay; siRNA knockdown in VLCAD-deficient fibroblasts with acylcarnitine profiling

    PMID:24158852

    Open questions at the time
    • Structural basis for how ACAD9 participates in assembly intermediates unknown
    • Direct binding partners in assembly not identified biochemically
  5. 2015 High

    ACAD9 knockout in HEK293 cells confirmed that both functions operate physiologically in the same cell, and systematic enzymatic characterization of 16 pathogenic mutations revealed that residual dehydrogenase activity inversely correlates with clinical severity.

    Evidence CRISPR/TALEN knockout with FAO and complex I rescue; prokaryotic expression and enzymatic assay of 16 patient mutations; genotype-phenotype correlation

    PMID:25721401

    Open questions at the time
    • Assembly-deficient vs. enzymatically-deficient mutation classes not functionally separated in patients
    • In vivo tissue-specific requirements not addressed
  6. 2021 High

    Reconstitution of the ACAD9–ECSIT–NDUFAF1 ternary complex and structural analysis defined the molecular architecture: ECSIT binds ACAD9's ETF-interaction site displacing FAD and abolishing enzymatic activity, explaining mutual exclusivity of the two functions; tissue-specific knockout mice confirmed that cardiac ACAD9 loss is lethal and that ACAD9 stabilizes ECSIT protein levels in vivo.

    Evidence In vitro ternary complex reconstitution with SAXS, mutagenesis, and FAD quantification; Cre-lox cardiac- and muscle-specific KO mice with survival and biochemical readouts

    PMID:34556413 PMID:34646991

    Open questions at the time
    • High-resolution atomic structure of the ternary complex not determined
    • How the ternary complex engages complex I assembly intermediates (e.g., the Q-module) not resolved
    • Mechanism by which ACAD9 stabilizes ECSIT at the protein level unclear
  7. 2025 Medium

    In cancer cells, ACAD9 loss redirects linoleic acid flux from β-oxidation to membrane lipid biosynthesis, increasing PUFA incorporation and sensitizing cells to ferroptosis — linking ACAD9's metabolic function to cancer cell vulnerability.

    Evidence In vivo genome-wide CRISPR screen in ovarian cancer; multi-omics and lipid flux analyses

    PMID:40618880

    Open questions at the time
    • Generalizability beyond ovarian cancer models not tested
    • Relative contributions of FAO loss vs. complex I dysfunction to ferroptosis sensitization not separated
    • In vivo therapeutic relevance not established

Open questions

Synthesis pass · forward-looking unresolved questions
  • A high-resolution structure of ACAD9 in complex with ECSIT and NDUFAF1, and understanding of how this ternary complex is recruited to and released from complex I assembly intermediates, remain key unresolved questions.
  • No cryo-EM or X-ray structure of the ACAD9–ECSIT–NDUFAF1 complex
  • Temporal ordering of ACAD9 engagement with complex I intermediates not defined
  • Regulatory switch controlling partitioning of ACAD9 between FAO and complex I assembly unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016491 oxidoreductase activity 4 GO:0044183 protein folding chaperone 3
Localization
GO:0005739 mitochondrion 4
Pathway
R-HSA-1852241 Organelle biogenesis and maintenance 5 R-HSA-1430728 Metabolism 4
Partners
ECSITNDUFAF1
Complex memberships
ACAD9-ECSIT-NDUFAF1 complex I assembly complex

Evidence

Reading pass · 12 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2002 ACAD9 encodes a mitochondrial acyl-CoA dehydrogenase with enzymatic dehydrogenase activity on long-chain fatty acyl-CoAs, demonstrated by in vitro enzymatic assay on palmitoyl-CoA (C16:0) and stearoyl-CoA (C18:0) using recombinant protein. Recombinant protein expression and in vitro enzymatic assay Biochemical and biophysical research communications High 12359260
2007 ACAD9 demonstrates maximum activity with unsaturated long-chain acyl-CoAs and functions in a distinct fatty acid oxidation pathway from VLCAD, as evidenced by lack of mutual compensation in patients deficient in either enzyme. Patient cell/biochemical studies; mRNA and protein defect characterization; substrate specificity analysis American journal of human genetics Medium 17564966
2010 ACAD9 has an essential role in mitochondrial respiratory chain complex I assembly; expression of wild-type ACAD9 corrects complex I deficiency in patient-derived fibroblasts, establishing a novel function beyond fatty acid oxidation. Complementation of patient fibroblasts with wild-type ACAD9; complex I activity rescue assay Nature genetics High 21057504
2010 A missense mutation (R532W) in ACAD9 causes complex I deficiency, and wild-type but not mutant ACAD9 restores complex I activity in patient fibroblasts, confirming the complex I assembly role of ACAD9; riboflavin supplementation improves complex I activity. Lentiviral transduction of wild-type and mutant ACAD9 into patient fibroblasts; complex I activity measurement Brain : a journal of neurology High 20929961
2013 ACAD9 displays fatty acid oxidation enzyme activity in vivo; knockdown of ACAD9 in VLCAD-deficient fibroblasts reveals ACAD9 is responsible for production of C14:1-carnitine from oleate and C12-carnitine from palmitate. siRNA knockdown in VLCAD-deficient fibroblasts; acylcarnitine profiling upon fatty acid loading Human molecular genetics High 24158852
2013 Catalytically inactive ACAD9 can partially to fully rescue complex I biogenesis in ACAD9-deficient cells and is incorporated into high-molecular-weight complex I assembly intermediates, demonstrating that enzymatic activity is not required for the complex I assembly function. Expression of catalytically inactive ACAD9 mutant in ACAD9-deficient cells; BN-PAGE analysis of assembly intermediates; complex I activity measurement Human molecular genetics High 24158852
2015 ACAD9 plays a physiological role in long-chain fatty acid oxidation in cells with high ACAD9 expression (HEK293 cells); ACAD9 knockout affects both long-chain fatty acid oxidation and complex I, both rescued by wild-type ACAD9; residual ACAD enzymatic activity of pathogenic mutations inversely correlates with clinical severity. ACAD9 knockout in HEK293 cells; fatty acid oxidation assays; prokaryotic expression system for enzymatic activity measurement of 16 mutations; correlation with patient phenotype Human molecular genetics High 25721401
2021 ACAD9 forms a core mitochondrial complex I assembly complex with ECSIT and NDUFAF1: ACAD9 binds the carboxy-terminal half of ECSIT, while NDUFAF1 binds the amino-terminal half of ECSIT; the ternary ACAD9/ECSIT/NDUFAF1 complex is soluble and stable whereas binary complexes are not. ECSIT binding at the ETF binding site in the amino-terminal domain of ACAD9 causes loss of FAD and enzymatic activity, demonstrating the two functions of ACAD9 are mutually exclusive. Protein-protein interaction studies (binary and ternary complex assembly); small-angle X-ray scattering (SAXS); molecular modeling; mutagenesis; FAD quantification; enzymatic activity assays iScience High 34646991
2021 Cardiac-specific ACAD9 knockout mice develop severe neonatal cardiomyopathy and die by 17 days of age with severe mitochondrial dysfunction; muscle-specific knockouts are viable but show muscle weakness. ECSIT protein levels are significantly reduced in the absence of ACAD9, consistent with ACAD9's role as a chaperone for ECSIT in complex I assembly. Cre-lox tissue-specific knockout mouse models; cardiac and muscle function assays; Western blot for ECSIT; mitochondrial function assays in vitro Molecular genetics and metabolism High 34556413
2013 ACAD9 functions as a complex I assembly protein; loss-of-function mutations reduce complex I holoprotein levels as shown by Western blot in muscle and fibroblasts, and the protein is a flavin adenine dinucleotide (FAD)-containing flavoprotein. Western blot quantification of complex I holoprotein; biochemical analysis of patient muscle and fibroblasts JAMA neurology Medium 23836383
2016 ACAD9 harbors a homodimer structure, and a p.Arg417Cys mutation creates an aberrant dimer as shown by protein modeling, contributing to loss of function. Protein structural modeling; protein expression analysis JIMD reports Low 26475292
2025 ACAD9 deficiency in ovarian cancer cells triggers mitochondrial respiratory collapse, ROS accumulation, and under linoleic acid-enriched conditions redirects LA flux from β-oxidation toward membrane lipid biosynthesis, increasing polyunsaturated fatty acid incorporation and sensitizing cells to ferroptosis. In vivo genome-wide CRISPR/Cas9 knockout screen; multi-omics integration; mechanistic cell biology assays for ROS, respiration, and lipid flux Cancer letters Medium 40618880

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 184 21057504
2007 A new genetic disorder in mitochondrial fatty acid beta-oxidation: ACAD9 deficiency. American journal of human genetics 97 17564966
2010 Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective ACAD9: new function for an old gene. Brain : a journal of neurology 92 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 55 30025539
2013 ACAD9, a complex I assembly factor with a moonlighting function in fatty acid oxidation deficiencies. Human molecular genetics 55 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 39 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 22 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 11 28070495
2021 Development and characterization of a mouse model for Acad9 deficiency. Molecular genetics and metabolism 10 34556413
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 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
2023 Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A). Frontiers in immunology 8 37388727
2017 Severe defect in mitochondrial complex I assembly with mitochondrial DNA deletions in ACAD9-deficient mild myopathy. Muscle & nerve 8 27438479
2025 Metabolic gatekeeper ACAD9 coordinates linoleic acid metabolism and redox homeostasis via mitochondrial complex I to drive ovarian cancer progression. Cancer letters 6 40618880
2022 Severe Antenatal Hypertrophic Cardiomyopathy Secondary to ACAD9-Related Mitochondrial Complex I Deficiency. Molecular syndromology 5 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 2 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 1 40806260