IadC (Vapar_1492) is the 330-amino-acid Class IA NADH:ferredoxin oxidoreductase component of the indole-3-acetic acid (IAA) Rieske non-heme iron dioxygenase system (IadD/IadE-IadC) in Variovorax paradoxus S110. Structurally, IadC folds into a triangle-shaped three-domain monomer comprising an N-terminal FMN-binding domain (residues 1-115), a central NADH-binding domain (residues 116-238), and a C-terminal plant-type [2Fe-2S] ferredoxin domain (residues 239-329 in the crystal structure PDB 7YLR; annotated as residues 245-330 by ProSite/UniProt) coordinated by four cysteine residues. IadC oxidizes NADH and transfers electrons via its non-covalently bound FMN cofactor and intramolecular [2Fe-2S] cluster to the Rieske [2Fe-2S] center of IadD, driving the aerobic dioxygenation of IAA to 2-oxindole-3-acetic acid (oxIAA) and enabling rapid auxin clearance and reversion of microbiome-induced root growth inhibition in the plant rhizosphere.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000120 | MODIFY | Summary: IadC is a Class IA NADH-dependent FMN/[2Fe-2S] reductase that transfers electrons from NADH to the Rieske [2Fe-2S] dioxygenase IadD/IadE. Reason: The broad term oxidoreductase activity (GO:0016491) is true but too general; X-ray crystallography (PDB 7YLR) and enzymatic reconstitution establish that IadC functions specifically as an NADH-dependent ferredoxin/Rieske dioxygenase reductase (GO:0008860). Proposed replacements: ferredoxin-NAD+ reductase activity Supporting Evidence: PMID:37459330 FMN is thought to mediate electron transfer from the NADH electron donor to the [2Fe-2S] electron acceptor, which further delivers the electrons to the dioxygenase for the catalysis reaction [29,30] (Fig 4C and 4E). |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | TOO GENERAL | Summary: IadC coordinates a [2Fe-2S] cluster in its C-terminal ferredoxin domain. Reason: The parent term iron-sulfur cluster binding (GO:0051536) is true of IadC but uninformative because its more specific child term 2 iron, 2 sulfur cluster binding (GO:0051537) is already accepted on this record and directly verified by the 1.68 A crystal structure (PDB 7YLR). Supporting Evidence: PMID:37459330 The [2Fe-2S] cluster is coordinated by 4 Cys residues in 2 peripheral loops, facing the center of the tri-lobbed IadC (Fig 4C, bottom panel). |
| GO:0051537 2 iron, 2 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: IadC coordinates a plant-type [2Fe-2S] cluster via four cysteine residues in its C-terminal domain (residues 239-329). Reason: Directly verified by the 1.68 A crystal structure of V. paradoxus S110 IadC (PDB 7YLR) and mutagenesis of Cys279. Supporting Evidence: PMID:37459330 The [2Fe-2S] cluster is coordinated by 4 Cys residues in 2 peripheral loops, facing the center of the tri-lobbed IadC (Fig 4C, bottom panel). |
| GO:0008860 ferredoxin-NAD+ reductase activity | IDA PMID:37459330 Structural and biochemical characterization of the key compo... | NEW | Summary: IadC catalyzes NADH oxidation and electron transfer via FMN and its intramolecular [2Fe-2S] ferredoxin domain to the Rieske [2Fe-2S] center of IadD. Reason: Crystal structure (PDB 7YLR) and in vitro enzymatic assays show that IadC is a Class IA FMN-type NADH:ferredoxin oxidoreductase required to supply electrons from NADH to IadD/IadE. Supporting Evidence: PMID:37459330 FMN is thought to mediate electron transfer from the NADH electron donor to the [2Fe-2S] electron acceptor, which further delivers the electrons to the dioxygenase for the catalysis reaction [29,30] (Fig 4C and 4E). |
| GO:0010181 FMN binding | IDA PMID:37459330 Structural and biochemical characterization of the key compo... | NEW | Summary: IadC binds an FMN cofactor at the interface of its FMN-binding and NADH-binding domains via His59, Phe233, Arg58, Arg89, and Ser92. Reason: Directly resolved in the 1.68 A crystal structure of V. paradoxus S110 IadC (PDB 7YLR) and validated by site-directed mutagenesis. Supporting Evidence: PMID:37459330 FMN resides at the interface of the FBD and NBD domains, with the isoalloxazine ring sandwiched between the side chains of His59 and Phe233 and anchored by polar contacts with nearby residues. Further, the ribityl moieties of FMN are stabilized by polar contacts with residues Arg58, Arg89, and Ser92 (Fig 4C, right panel). |
| GO:0042437 indoleacetic acid catabolic process | IDA PMID:37459330 Structural and biochemical characterization of the key compo... | NEW | Summary: IadC supplies the reducing equivalents required by IadD/IadE to oxygenate indole-3-acetic acid (IAA) to 2-oxindole-3-acetic acid (oxIAA). Reason: In vitro reconstitution (PMID:37459330) and in vivo complementation in Variovorax (PMID:36266335) demonstrate that IadC functions in the initial step of IAA catabolism. Supporting Evidence: PMID:37459330 Together, these data suggest that IAA was converted to oxIAA by the dioxygenase system composed of the IadD/E dioxygenase and the IadC reductase (Fig 5E). PMID:36266335 However, only the CL14 ΞHS33 pBBR1::iadCDE strain containing all three genes was able to revert the Arthrobacter CL28-dependent RGI to CL14 wild-type levels. |
| GO:0005829 cytosol | IC PMID:37459330 Structural and biochemical characterization of the key compo... | NEW | Summary: IadC is a soluble monomeric reductase in the bacterial cytosol. Reason: Inferred by curator (IC) because IadC is a soluble cytoplasmic enzyme lacking signal peptides or transmembrane helices that partners with the cytosolic IadD/IadE complex to process imported IAA. Supporting Evidence: PMID:37459330 IAA transported to the bacterial cell would be first processed by the iadd/E-iadc dioxygenase system and converted to oxiaa in the presence of molecular oxygen, thereby deactivating the auxin IAA. |
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Download this section (compressed HTML)Q: Which endogenous Variovorax or E. coli cellular reductases can partially substitute for IadC when iadDE is expressed in the absence of iadC?
Experiment: Measure the midpoint reduction potentials of the FMN and [2Fe-2S] cofactors in V. paradoxus S110 IadC by spectroelectrochemical titration and determine the rate constant of inter-protein electron transfer to IadD/IadE.
Hypothesis: The domain orientation of the FeSD domain against the NBD domain in IadC tunes the [2Fe-2S] redox potential for rapid, thermodynamically favorable electron delivery to the Rieske cluster of IadD.
Type: Spectroelectrochemistry and stopped-flow kinetics
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