Fig 1.
IadK2 encoded by the Variovorax iad operon selectively binds with auxin IAA.
(A) Schematics for the auxin IAA-degradation (iad) operon in Variovorax paradoxus CL14. Ten genes ranging from IMG gene ids 2643613661–2643613670 are illustrated. (B) Chemical structures of IAA and auxin analogs. IAA is the most dominant natural auxin in most plants. Other endogenous or synthetic auxins or analogs such as 4-Cl-IAA, IBA, IPA, NAA, and 2,4-D are also displayed. (C) ITC measurement of IAA binding to iadk2. The binding isotherm for iadk2-IAA is biphasic, revealing 2 dissociation constants. Three independent measurements were performed and the Kd values are shown as mean ± SEM. (D) ITC measurements of auxin analogs to iadk2. The binding of other auxins and analogs to iadk2 is distinct from IAA with a single-phase isotherm and substantially reduced binding affinity. ND: no binding detected. Kd values are shown as mean ± SEM from 3 or more independent measurements. (E) TSA for iadk2 with IAA and analogs. The orthogonal TSA experiments were performed for iadk2 with different auxins and analogs to further determine the binding specificity. Three replicates of each TSA experiments were performed. Error bars represent the standard deviations. Source data for C–E can be found in S1 Data. IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; IPA, indole-3-propionic acid; ITC, isothermal titration calorimetry; NAA, 1-naphthylacetic acid; TSA, thermal shift assay; 4-Cl-IAA, 4-chloroindole-3-acetic acid.
Fig 2.
Structure of IadK2 and the IAA-binding pocket.
(A) Domain organization of iadk2. Iadk2 is composed of 2 domains, Domain 1 and Domain 2. (B) Crystal structure of iadk2. Iadk2 features a two-lobed structure with Domain1 and Domain 2 connected by a linker region. L1–L3 indicate the 3 linkers connecting the 2 lobes of iadk2. Such overall structural features and that L2 contains 2 α helices group iadk2 to the B-III subcluster of sbps. The same color scheme in A is applied. (C) Structure of iadk2 complexed with IAA calculated from docking and MD simulation. A similar substrate pocket at the interface of the 2 lobes of iadk2 was predicted, resembling other sbps. The right panel displays the calculated IAA-binding pocket. Iadk2 is colored in gray and IAA is shown as yellow sticks. Key residues potentially involved in IAA binding are shown in sticks representation. (D) ITC measurements of IAA binding to iadk2 mutants. ND: no binding detected. Weak binding: KD > 100 μM. Kd values are shown as mean ± SD from 3 or more independent measurements. (E) TSA for iadk2 mutants and IAA. Three replications of each TSA experiments were performed. Data are presented as mean ± SEM. Source data for D and E can be found in S1 Data. IAA, indole-3-acetic acid; ITC, isothermal titration calorimetry; MD, molecular dynamics; TSA, thermal shift assay.
Fig 3.
Characterization of the IadD/Enon-heme Rieske dioxygenase complex.
(A) ITC measurement of IAA binding to the E complex. Kd values are shown as mean ± SEM from 3 or more independent measurements. (B) Crystal structure of iadd/E. Upper panel: Domain organization of iade and iadd. Bottom: one heterodimer of iadd and iade in the asymmetric unit. Iadd is composed of 2 domains. The catalytic domain of iadd is colored in light blue and the Rieske domain is colored in pale cyan. Positions of the ferrous iron in the catalytic domain and the [2Fe-2S] cluster in the Rieske domain are indicated. The iade is a single domain protein and is colored in pale green. (C) The 2.6 Å cryo-EM density of the iadd/E complexed with IAA. A mushroom-shaped, heterohexameric structure was resolved. The top and side views are displayed. (D) Overlay of the apo and IAA-bound structures of iadd/E. The IAA-bound heterohexamer of iadd/E (colored by subunits) was superimposed with that of the apo state generated with crystal symmetry-equivalent copies (colored in gray). No significant conformational changes were identified except some regions at the crystal-packing interfaces. The inset panel shows a zoom-in view of the active site composed of the [2Fe-2S] cluster, the mononuclear iron and the substrate-binding pocket. (E) The IAA-binding pocket in iadd. The indole ring of IAA and the mononuclear iron are in relatively perpendicular orientation. Key residues in the IAA-binding pocket are shown as sticks. (F) ITC measurements of IAA binding to iadd mutants. Mutations of residues in the IAA-binding pocket as shown in E disrupted the binding. ND: no binding detected. Source data for A and F can be found in S1 Data. IAA, indole-3-acetic acid; ITC, isothermal titration calorimetry.
Fig 4.
Reductase IadC and dioxygenase IadD/E catalyze IAA transformation.
(A) IAA transformation assay. Purified iadd/E and iadc were used to test the in vitro IAA transformation. The amounts of IAA were quantified using the Salkowski reagent and adsorption at 530 nm. Data are presented as mean ± SEM. (B) Time and concentration dependence of IAA transformation by iadc and iadd/E. IAA mixed with the Salkowski reagent show pink color, which turned colorless after treatment with iadc and iadd/E. Top panels show the color change of IAA reaction mixtures in the presence of Salkowski reagent over time (left, 0.7 μM iadc/D/E enzymes) or concentrations of iadc/D/E (right, reacted for 30 min), from pink to colorless. Below graphs illustrate the changes of quantified IAA amounts (adsorption at 530 nm) over time (left) or enzyme concentrations (right). (C) The determined crystal structure of iadc. Iadc possesses a tri-lobed structure, consisting of 3 domains including FBD, NBD, and fesd. The [2FE-2S]-binding pocket in fesd and the FMN-binding pocket in FBD are displayed, with key residues participated in the binding indicated. (D) Effects of iadc mutations on IAA transformation. In vitro IAA transformation assay was performed with both WT and mutated iadc in the presence of iadd/E. Data are presented as mean ± SEM. (E) Schematic illustration of the functional cooperation of iadc and the iadd/E. Iadc transfers electron to the active site of iadd composed of [2Fe-2S] and ferrous iron to catalyze IAA transformation. The inset panel displays key residues in iadd surrounding the [2Fe-2S] cluster and the ferrous iron, potentially essential for the electron transfer. (F) Effects of iadd mutations on IAA transformation. Both WT iadd/E and mutants defective in substrate binding or electron transfer were tested with the in vitro IAA degradation assay. Data are presented as mean ± SEM. Source data for A, B, D, and F can be found in S1 Data. FBD, FMN-binding domain; IAA, indole-3-acetic acid; NBD, NADH-binding domain; WT, wild type.
Fig 5.
The IadD/E-IadCdioxygenase-reductase system converted IAA to oxIAA.
(A) HPLC analysis of IAA before (in pink) and after transformation (in blue) by iadd/E-iadc. (B) HRMS analysis of the transformed products of IAA by iadd/E-iadc. The elution for potential product peak in HPLC was collected and analyzed by HRMS. Characteristic peaks for oxiaa were identified, which agreed with the calculated mass. Source data for A and B can be found in S1 Data. (C) Color development of IAA and analogs in Salkowski reagent. The listed compounds (0.5 mm) developed different colors after mixing with the Salkowski reagent. (D) The absorbance spectra of IAA, oxiaa, and the products by iadd/E-iadc. The spectrum scan for IAA, oxiaa, and transformed IAA by the iadd/E-iadcafter mixing with the Salkowski reagent was performed. (E) The mechanism of IAA transformation by iadd/E-iadc. The iadd/E-iadcdioxygenase system catalyzes the incorporation of oxygen atoms to IAA from O2, leading to the formation of 2-hydroxyindole-3-acetic acid, which potentially primarily exist as the stable “keto” form, oxiaa. HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; IAA, indole-3-acetic acid.
Fig 6.
Ectopic expression of Variovorax genes iadC/D/E enables bacterial transformation of IAA by E. coli.
(A) Schematic for the design of the experiment for the bacterial IAA transformation assay. Vectors containing the indicated iad genes were transformed to E. Coli and the transformation of IAA in the LB medium was monitored using the Salkowski reagent. (B) In vivo IAA transformation assay for E. coli containing different iad genes as indicated in A. Little IAA transformation was observed when the empty vector carrying no iad genes was transformed to E. coli. The presence of all 3 genes iadc/D/E resulted in efficient IAA transformation. (C) Effects of Iad proteins mutants in the in vivo IAA transformation by E. coli. Loss-of-function mutations of iadk2 (S104V/W172L), iadd (H216A), and iadc (C279S) were tested. Minimal IAA transformation occurred with the empty vector or in the absence of protein expression inducer IPTG. Source data for B and C can be found in S1 Data. (D) Schematic diagram of IAA transformation by the Variovorax iad operon. 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. Despite not required for the engineered E. coli, iadk2 may mediate the efficient IAA uptake by Variovorax from the environment. However, further studies would be required to examine whether iadk2 is need for Variovorax IAA degradation. IAA, indole-3-acetic acid; IPTG, isopropyl-β-D-thiogalactoside.