FLAD1 encodes human FAD synthase (FMN adenylyltransferase; FMNAT; EC 2.7.7.2), the bifunctional, multidomain enzyme that catalyzes the final, committed step of FAD cofactor biosynthesis: adenylylation of flavin mononucleotide (FMN) with ATP to give flavin adenine dinucleotide (FAD) plus diphosphate, a reaction that requires a divalent metal (optimally Mg2+). FAD is the cofactor of hundreds of flavoenzymes, including respiratory-chain complexes I and II, the acyl-CoA dehydrogenases of fatty-acid beta-oxidation, MTHFR, dimethylglycine dehydrogenase, and the histone demethylase LSD1/KDM1A, so FLAD1 is central to cellular energy metabolism and redox biology. The C-terminal PAPS-reductase-family domain carries the FMN adenylyltransferase (FAD synthase) activity, while an N-terminal molybdopterin-binding-resembling (MPTb) domain together with a KH domain confers a FAD hydrolase / FAD diphosphatase activity (EC 3.6.1.18) and a weaker NADH diphosphatase activity (EC 3.6.1.22), allowing the enzyme to control the FAD/FMN balance. Beyond catalysis, the protein binds its FAD product tightly and acts as a FAD chaperone, delivering the cofactor directly to client apo-flavoproteins. The enzyme functions as a C2-symmetric homodimer. Alternative splicing of FLAD1 produces isoforms that differ in the N-terminal region and in subcellular targeting: the longer isoform 1 bears a mitochondrial transit peptide and localizes to the mitochondrial matrix, the shorter isoform 2 is cytosolic and most abundant, and FADS is also detected in the nucleus. Biallelic loss-of-function variants cause lipid storage myopathy with combined respiratory-chain and multiple acyl-CoA dehydrogenase deficiency, a subset of which is riboflavin-responsive.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003919 FMN adenylyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) call for the core molecular function of FLAD1, the FMN adenylyltransferase (FAD synthase) that makes FAD from FMN and ATP. Correct and strongly supported by direct experimental data in human FLAD1. Supporting Evidence: PMID:16643857 FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. |
| GO:0006747 FAD biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of the biological process. FLAD1 carries out the final step of FAD biosynthesis (FAD from FMN, step 1/1), so this is the correct core process term. Supporting Evidence: PMID:25954742 the enzyme responsible for FMN adenylation to FAD. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Uninformative root-level catalytic activity term from an InterPro2GO mapping. FLAD1 has specific, well-characterized catalytic activities (FMN adenylyltransferase, FAD diphosphatase), so this generic term is an over-annotation superseded by the specific MF terms. |
| GO:0003919 FMN adenylyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (InterPro/RHEA/EC 2.7.7.2) assignment of the core FMN adenylyltransferase activity. Consistent with the direct experimental annotations; accept. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic subcellular-location mapping to nucleus. Nuclear localization of FADS is experimentally supported (a nuclear flavin pool supplying LSD1/KDM1A), so this is acceptable but non-core relative to the enzyme's cytosolic/mitochondrial catalytic roles. |
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA electronic annotation to the generic parent 'cytoplasm'. The specific cytosol localization (isoform 2) is experimentally established; this generic parent is redundant with the more precise GO:0005829 cytosol IDA annotation. |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic assignment to mitochondrial matrix. Concordant with experimental evidence that isoform 1 (with its N-terminal transit peptide) localizes to the mitochondrial matrix; accept but non-core. |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic subcellular-location mapping to cytosol. Cytosol is the location of the abundant isoform 2 and is directly supported experimentally; redundant with the IDA cytosol annotations. |
| GO:0006747 FAD biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (InterPro/UniPathway) assignment of the FAD biosynthetic process. Correct core process; redundant with the IBA and IDA annotations to the same term. |
| GO:0035529 NADH pyrophosphatase activity | IEA GO_REF:0000116 | KEEP AS NON CORE | Summary: Electronic RHEA mapping (RHEA:48868; EC 3.6.1.22) of a genuine but minor side activity of the N-terminal hydrolase domain. Experimentally demonstrated (isoform 2 hydrolyzes NADH and other ADP-containing dinucleotides), but this is a low-efficiency accessory activity, not a core function. Supporting Evidence: file:human/FLAD1/FLAD1-uniprot.txt FAD diphosphatase |
| GO:0047884 FAD diphosphatase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (RHEA:13889; EC 3.6.1.18) assignment of the FAD hydrolase / FAD diphosphatase activity. This is a real, experimentally verified second catalytic activity residing in the N-terminal MPTb/KH domain; accept. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: Bare 'protein binding' from a high-throughput interactome map (HuRI). Uninformative as a molecular function; the physiologically meaningful interactions of FADS are with its apo-flavoprotein clients (KDM1A, DMGDH), captured separately as the FAD chaperone/delivery role. Retained (per policy, not removed) but flagged as an over-annotation. |
| GO:0005515 protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | MARK AS OVER ANNOTATED | Summary: Bare 'protein binding' from a large-scale interaction-perturbation screen. Uninformative molecular-function term; retained but marked as over-annotated. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | MARK AS OVER ANNOTATED | Summary: Bare 'protein binding' from a proteome-scale interactome (BioPlex) dataset. Uninformative; retained but marked as over-annotated. |
| GO:0005515 protein binding | IPI PMID:40205054 Multimodal cell maps as a foundation for structural and func... | MARK AS OVER ANNOTATED | Summary: Bare 'protein binding' from a multimodal cell-map / functional-genomics interaction dataset. Uninformative molecular-function term; retained but marked as over-annotated. |
| GO:0042802 identical protein binding | IPI PMID:16189514 Towards a proteome-scale map of the human protein-protein in... | KEEP AS NON CORE | Summary: Homodimerization (self-interaction). Consistent with the established C2-symmetric homodimer of FADS, so biologically real, but self-association is a structural property rather than the enzyme's core catalytic function. |
| GO:0042802 identical protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | KEEP AS NON CORE | Summary: Self-interaction supporting the FADS homodimer (also documented structurally in the crystal structure). Biologically supported but non-core. Supporting Evidence: PMID:38688286 hFADS2 associates in a stable C2-symmetric dimer |
| GO:0042802 identical protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | KEEP AS NON CORE | Summary: Self-interaction consistent with the physiological homodimer. Retained as non-core. |
| GO:0006771 riboflavin metabolic process | TAS Reactome:R-HSA-196843 | KEEP AS NON CORE | Summary: Reactome-traceable assignment to riboflavin (vitamin B2) metabolism. FAD synthesis is the terminal branch of the riboflavin-to-FAD pathway, so this parent process is correct, though the more specific GO:0006747 FAD biosynthetic process better captures the direct role. |
| GO:0003919 FMN adenylyltransferase activity | TAS Reactome:R-HSA-196929 | ACCEPT | Summary: Reactome traceable annotation of the core FMN adenylyltransferase reaction (FLAD1 adenylylates FMN). Correct core molecular function. |
| GO:0005829 cytosol | IDA GO_REF:0000052 | ACCEPT | Summary: Direct immunofluorescence evidence (HPA) for cytosolic localization, matching the cytosolic isoform 2. Correct localization. |
| GO:0003919 FMN adenylyltransferase activity | EXP PMID:16643857 Over-expression in Escherichia coli and characterization of ... | ACCEPT | Summary: Direct enzymatic characterization of recombinant human FADS isoforms 1 and 2, both of which had FMN adenylyltransferase (FAD synthase) activity with a strict Mg2+ requirement. Strong experimental support for the core function. Supporting Evidence: PMID:16643857 Both isoforms possessed FADS activity and had a strict requirement for MgCl(2) |
| GO:0003919 FMN adenylyltransferase activity | EXP PMID:21924249 The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavi... | ACCEPT | Summary: Human FAD synthetase assayed for FMN adenylyltransferase activity, shown to accept RoFMN and synthesize RoFAD (substrate-scope study), confirming the core activity. Supporting Evidence: PMID:21924249 Consequently, roseoflavin adenine dinucleotide (RoFAD) was synthesized by the latter enzyme |
| GO:0003919 FMN adenylyltransferase activity | EXP PMID:23443125 Bacterial over-expression and purification of the 3'phosphoa... | ACCEPT | Summary: The isolated C-terminal PAPS-reductase domain of human FADS catalyzes FAD synthesis from ATP and FMN, directly localizing the FMN adenylyltransferase activity to that domain. Strong experimental support. Supporting Evidence: PMID:23443125 the PAPS reductase domain, per se, is able to catalyze the typical reactions of the FADS: FAD synthesis and its cleavage. |
| GO:0003919 FMN adenylyltransferase activity | EXP PMID:25135855 Significance of redox-active cysteines in human FAD synthase... | ACCEPT | Summary: Mutagenesis/kinetic study of redox-active cysteines in the FAD-synthesizing domain, measuring FAD synthesis rate directly. Confirms the core FMN adenylyltransferase activity. Supporting Evidence: PMID:25135855 FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. |
| GO:0035529 NADH pyrophosphatase activity | EXP PMID:31351152 The hidden side of the human FAD synthase 2. | KEEP AS NON CORE | Summary: Experimental characterization of the N-terminal hydrolase (FADHy) domain, which hydrolyzes FAD and, at lower efficiency, NADH (an ADP-containing dinucleotide). A genuine minor side activity; not the enzyme's core function. Supporting Evidence: PMID:31351152 Reduced glutathione stimulated whereas NADH inhibited the hydrolytic activity. |
| GO:0047884 FAD diphosphatase activity | IDA PMID:26277395 Human FAD synthase is a bi-functional enzyme with a FAD hydr... | ACCEPT | Summary: Direct demonstration that human FADS hydrolyzes FAD (to FMN + AMP) via its N-terminal molybdopterin-binding domain, in a Co2+/mersalyl-dependent reaction. This establishes the second, genuine catalytic activity of the bifunctional enzyme. Supporting Evidence: PMID:26277395 The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. |
| GO:0047884 FAD diphosphatase activity | IDA PMID:31351152 The hidden side of the human FAD synthase 2. | ACCEPT | Summary: Detailed kinetic characterization of the FAD hydrolytic activity of isoform 2 under near-physiological conditions (Co2+/K+ stimulated), confirming the FAD diphosphatase activity of the MPTb/FADHy domain. Supporting Evidence: PMID:31351152 Co2+ induced FAD hydrolysis was strongly stimulated in the presence of K+, reaching a Vmax higher than that of FAD synthesis. |
| GO:0003919 FMN adenylyltransferase activity | IDA PMID:25954742 Remaining challenges in cellular flavin cofactor homeostasis... | ACCEPT | Summary: Direct assay of recombinant isoform 2 FAD synthesis (ordered bi-bi mechanism, ATP binding before FMN). Confirms core FMN adenylyltransferase activity and additionally demonstrates the FAD-chaperone delivery to client apo-flavoproteins. Supporting Evidence: PMID:25954742 FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. |
| GO:0006747 FAD biosynthetic process | IDA PMID:25954742 Remaining challenges in cellular flavin cofactor homeostasis... | ACCEPT | Summary: Direct evidence linking FADS activity to cellular FAD synthesis and delivery. Correct core biological process. Supporting Evidence: PMID:25954742 the enzyme responsible for FMN adenylation to FAD. |
| GO:0005634 nucleus | IDA PMID:25954742 Remaining challenges in cellular flavin cofactor homeostasis... | KEEP AS NON CORE | Summary: Direct (confocal immunofluorescence) evidence for a nuclear pool of FADS, consistent with FAD supply to the nuclear demethylase LSD1/KDM1A. Real localization but non-core relative to the enzyme's principal cytosolic/mitochondrial function. Supporting Evidence: PMID:25954742 a novel FADS localisation was found to be the nucleus of mammalian cells |
| GO:0005829 cytosol | IDA PMID:25954742 Remaining challenges in cellular flavin cofactor homeostasis... | ACCEPT | Summary: Direct evidence for cytosolic localization of isoform 2, the most abundant FADS isoform. Correct localization. Supporting Evidence: PMID:25954742 Isoform 1 and 2 are located in mitochondria and cytosol, respectively |
| GO:0003919 FMN adenylyltransferase activity | IDA PMID:21951714 Human FAD synthase (isoform 2): a component of the machinery... | ACCEPT | Summary: Direct enzymatic characterization of purified recombinant isoform 2 catalyzing FAD assembly from ATP and FMN, with FAD release as the rate-limiting step. Strong support for the core function. Supporting Evidence: PMID:21951714 the enzyme catalyzes FAD assembly from ATP and FMN |
| GO:0003919 FMN adenylyltransferase activity | IDA PMID:38688286 Structural insights into the bifunctional enzyme human FAD s... | ACCEPT | Summary: Crystal structure and functional dissection confirming that the C-terminal PAPS domain binds FAD and catalyzes its synthesis (FMN adenylyltransferase). Definitive structural and functional support for the core activity. Supporting Evidence: PMID:38688286 the C-terminal domain tightly binds FAD and catalyzes its synthesis |
| GO:0047884 FAD diphosphatase activity | IDA PMID:38688286 Structural insights into the bifunctional enzyme human FAD s... | ACCEPT | Summary: Structural and functional evidence that the N-terminal molybdopterin-binding plus KH domains form the minimal substructure hydrolyzing FAD (and other ADP-containing dinucleotides), assigning the FAD diphosphatase activity to that region. Supporting Evidence: PMID:38688286 the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. |
| GO:0005634 nucleus | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Sequence-similarity (ISS) transfer of nuclear localization from a rat ortholog. Concordant with the direct human nuclear-localization evidence; non-core. |
| GO:0003919 FMN adenylyltransferase activity | IDA PMID:20060505 Mitochondrial localization of human FAD synthetase isoform 1... | ACCEPT | Summary: Recombinant human FADS isoform 1 characterized enzymatically (Km/Vmax for FMN), confirming the core FMN adenylyltransferase activity for the mitochondrial isoform. Supporting Evidence: PMID:20060505 The recombinant hFADS1 produced in Escherichia coli showed apparent K(m) and V(max) values for FMN |
| GO:0005759 mitochondrial matrix | IDA PMID:20060505 Mitochondrial localization of human FAD synthetase isoform 1... | KEEP AS NON CORE | Summary: Direct evidence (import assays and immunofluorescence) that isoform 1, but not isoform 2, localizes to mitochondria; the N-terminal transit peptide targets the matrix. Genuine isoform-specific localization; non-core relative to the dominant cytosolic FADS. Supporting Evidence: PMID:20060505 hFADS1, but not hFADS2, localizes in mitochondria |
| GO:0006747 FAD biosynthetic process | IDA PMID:20060505 Mitochondrial localization of human FAD synthetase isoform 1... | ACCEPT | Summary: Direct evidence that the mitochondrial isoform 1 carries out FAD synthesis, supporting the core FAD biosynthetic process in the mitochondrial compartment. Supporting Evidence: PMID:20060505 FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | KEEP AS NON CORE | Summary: High-throughput mitochondrial-proteome detection of FLAD1. Consistent with the experimentally established mitochondrial localization of isoform 1; the more specific mitochondrial matrix term is better supported by IDA. Non-core. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-196929 | ACCEPT | Summary: Reactome traceable assignment of cytosol as the compartment for the FMN adenylyltransferase reaction, matching the cytosolic isoform 2. Correct localization. |
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Download this section (compressed HTML)Q: Is the FAD diphosphatase / NADH diphosphatase activity of the N-terminal domain physiologically significant in vivo, or primarily a regulatory switch controlling the local FAD/FMN and FAD/NAD balance?
Experiment: Compartment-specific flavin metabolomics in FLAD1 isoform-knockout/knock-in cells to quantify the in vivo contribution of the FAD synthase versus FAD hydrolase activities to mitochondrial, cytosolic, and nuclear FAD pools.
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