IadD (Vapar_1493) is the 437-amino-acid catalytic alpha subunit of the heterohexameric (alpha3beta3) indole-3-acetic acid (IAA) Rieske non-heme iron aromatic-ring-hydroxylating dioxygenase complex (IadD/IadE) in Variovorax paradoxus S110. Together with the structural beta subunit IadE and the FMN/[2Fe-2S]-containing NADH-dependent reductase IadC, IadD catalyzes the initial aerobic oxygenation of the plant hormone indole-3-acetic acid (IAA) into 2-hydroxyindole-3-acetic acid, which tautomerizes to biologically inactive 2-oxindole-3-acetic acid (oxIAA) and is further degraded via dioxindole-3-acetic acid and isatin to anthranilic acid. Structurally, each IadD monomer comprises an N-terminal Rieske domain (residues 44-160) that coordinates a [2Fe-2S] cluster via Cys85, His87, Cys106, and His109, and a C-terminal catalytic domain (residues 1-43 and 161-437) that coordinates a mononuclear ferrous iron center via the 2-His-1-carboxylate facial triad His216, His221, and Asp377 adjacent to the IAA substrate-binding pocket. In the rhizosphere, IAA catabolism by the IadD/IadE-IadC dioxygenase system enables Variovorax to buffer root-zone auxin levels and prevent auxin-induced root growth inhibition in host plants.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: IadD coordinates a catalytic mononuclear ferrous iron ion at its active site via His216, His221, and Asp377. Reason: High-resolution crystal (PDB 7YLS) and cryo-EM (PDB 8H2T) structures of Variovorax paradoxus S110 IadD/IadE directly resolve the catalytic mononuclear iron ion in the C-terminal domain of IadD coordinated by His216, His221, and Asp377. Supporting Evidence: PMID:37459330 The [2Fe-2S] is stabilized by contacts with Cys85, His87, Cys106, and His109 and the mononuclear iron coordinates with His216, His221, and Asp377. |
| GO:0051537 2 iron, 2 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: IadD binds a Rieske-type [2Fe-2S] cluster in its N-terminal Rieske domain (residues 44-160) via Cys85, His87, Cys106, and His109. Reason: Directly verified by X-ray crystallography (PDB 7YLS) and cryo-EM (PDB 8H2T) of V. paradoxus S110 IadD/IadE, showing that Cys85, His87, Cys106, and His109 coordinate the Rieske [2Fe-2S] cluster. Supporting Evidence: PMID:37459330 The [2Fe-2S] is stabilized by contacts with Cys85, His87, Cys106, and His109 and the mononuclear iron coordinates with His216, His221, and Asp377. |
| GO:0016708 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of two atoms of oxygen into one donor | IDA PMID:37459330 Structural and biochemical characterization of the key compo... | NEW | Summary: IadD is the catalytic alpha subunit of the Rieske non-heme iron aromatic-ring-hydroxylating dioxygenase complex (IadD/IadE) that oxygenates indole-3-acetic acid (IAA) using electrons supplied from NADH via IadC. Reason: Purified V. paradoxus S110 IadD/IadE and IadC reconstitute NADH- and O2-dependent dioxygenation of IAA to 2-oxindole-3-acetic acid (oxIAA) in vitro, and active-site mutations in IadD abolish activity. Supporting Evidence: PMID:37459330 Indeed, IAA was transformed efficiently in the presence of both IadD/E and IadC, whereas neither IadD/E nor IadC alone could catalyze the transformation (Fig 4A and 4B), suggesting a potential two-component dioxygenase system for IAA conversion. 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). |
| GO:0042437 indoleacetic acid catabolic process | IMP PMID:36266335 Diverse MarR bacterial regulators of auxin catabolism in the... | NEW | Summary: IadD catalyzes the first committed step of aerobic indole-3-acetic acid (IAA) catabolism in Variovorax. Reason: Both genetic deletion/complementation in Variovorax and E. coli (PMID:36266335) and in vitro enzymatic reconstitution with V. paradoxus S110 IadD/IadE and IadC (PMID:37459330) establish that IadD is required for IAA catabolism. Supporting Evidence: PMID:36266335 To test the necessity of these genes, we constructed knockout strains CL14 ΞiadDE and CL14 ΞiadCDE. Both lacked the ability to degrade IAA (Fig. 1d), demonstrating that iadD and iadE are necessary and sufficient for IAA degradation by Variovorax. 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). |
| GO:0005829 cytosol | IC PMID:37459330 Structural and biochemical characterization of the key compo... | NEW | Summary: IadD functions in the bacterial cytosol as part of the soluble alpha3beta3 IadD/IadE dioxygenase complex. Reason: Inferred by curator (IC) from the purification of soluble IadD/IadE heterohexamers lacking signal peptides or transmembrane helices and its intracellular function in oxygenating IAA imported into the bacterial cell. 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: What is the exact chemical mechanism by which the initial oxygenation product of IadD/IadE (2-hydroxyindole-3-acetic acid / oxIAA) is further oxidized to dioxindole-3-acetic acid (DOAA) and cleaved to isatin by downstream enzymes in the iad operon?
Experiment: Perform single-turnover stopped-flow spectroscopy and cryo-EM of the transient IadC-IadD/IadE electron-transfer complex to map the docking interface between the IadC plant-type [2Fe-2S] domain and the IadD Rieske [2Fe-2S] domain.
Hypothesis: IadC docks transiently near the subunit interface of the IadD trimer to place its [2Fe-2S] cluster within electron-tunneling distance of the IadD Rieske [2Fe-2S] cluster.
Type: Cryo-EM and stopped-flow rapid kinetics
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