IIL1 (IPMI LSU1, LeuC; At4g13430) is the single large subunit of the plastid-localized, bacterial-type heterodimeric isopropylmalate isomerase (IPMI) of Arabidopsis. A member of the aconitase superfamily, it carries aconitase domains 1-3 and the [4Fe-4S] cluster, while one of three small subunits (SSU1-3, LeuD) supplies domain 4 and determines substrate specificity. The heterodimer catalyzes the reversible dehydration/rehydration that converts a 2-alkylmalate to the corresponding 3-alkylmalate via an alkylmaleate intermediate. Paired with SSU1, IIL1 isomerizes 2-isopropylmalate in L-leucine biosynthesis; paired with SSU2 or SSU3, it isomerizes 2-(omega-methylthio)alkylmalates in each cycle of methionine chain elongation, the first phase of aliphatic glucosinolate biosynthesis. Consistent with this dual role, reduction of IIL1 causes accumulation of 2-isopropylmalate and methylthioalkylmalate intermediates, lower levels of long-chain aliphatic glucosinolates and increased short-chain (C3) glucosinolates. IIL1 is expressed mainly in and along the vasculature and is located in the chloroplast stroma. A triplet-repeat expansion in an IIL1 intron underlies the temperature-dependent growth defect of accession Bur-0.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003861 3-isopropylmalate dehydratase activity | EXP PMID:19597944 Arabidopsis thaliana encodes a bacterial-type heterodimeric ... | ACCEPT | Summary: IIL1 (IPMI LSU1, LeuC) is the sole large subunit of the plastid heterodimeric isopropylmalate isomerase. Knockdown/knockout lines accumulate the IPMI substrate 2-isopropylmalate, showing the large subunit is required for isomerization of 2-IPM in Leu biosynthesis. Reason: The functional activity is enabled by a LeuC:LeuD heterodimer: IIL1 supplies domains 1–3 and the catalytic iron-sulfur site, while the small subunit supplies domain 4. Retain the supported activity claim, preserve the machine-sourced qualifier, and represent the protein contribution explicitly in core_functions.contributes_to_molecular_function. Possession of the catalytic cluster does not establish autonomous activity. Supporting Evidence: PMID:19597944 The IPMI substrate, 2-IPM, accumulates when transcript of IPMI LSU1 is reduced sufficiently. PMID:19597944 Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. PMID:19597944 However, in IPMIs of bacteria, archaea and plants domains 1–3 are found in a large subunit, while domain 4 is found in a small subunit. PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0003861 3-isopropylmalate dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of EC 4.2.1.33 / RHEA:32287 from the IPMI large-subunit InterPro signature; consistent with experimental data. Reason: Agrees with the experimental EXP annotation from Knill et al. 2009 and He et al. 2010. Supporting Evidence: PMID:19597944 The IPMI substrate, 2-IPM, accumulates when transcript of IPMI LSU1 is reduced sufficiently. |
| GO:0008652 amino acid biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO assignment of the generic amino acid biosynthetic process from the LeuC/IPMI large-subunit family signature. Reason: Refine the general amino acid biosynthetic process to its supported L-leucine biosynthesis descendant, already annotated and supported by IPM accumulation in lsu1 mutants. Proposed replacements: L-leucine biosynthetic process Supporting Evidence: PMID:19597944 Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. |
| GO:0009098 L-leucine biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway mapping (L-leucine biosynthesis step 2/4) to IIL1. Reason: Experimentally supported: IIL1 is the only IPMI large subunit in Arabidopsis and lsu1 mutants accumulate 2-IPM, the Leu-pathway substrate of IPMI. Leu biosynthesis is one of the two core processes of this dual-function subunit (the other is Met chain elongation for aliphatic glucosinolates). Supporting Evidence: PMID:19597944 The IPMI substrate, 2-IPM, accumulates when transcript of IPMI LSU1 is reduced sufficiently. PMID:19597944 The genome contains a single gene for the large subunit (At4g13430, IPMI LSU1) and three genes for the small subunits of this enzyme PMID:33568694 It has been demonstrated that LeuC plays dual roles in both GLS and Leu biosynthesis |
| GO:0009507 chloroplast | HDA PMID:18431481 Sorting signals, N-terminal modifications and abundance of t... | MODIFY | Summary: Detected by MS in purified Arabidopsis chloroplasts (Zybailov et al. 2008). Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:18431481 Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. PMID:19597944 In Arabidopsis the latter protein has also been detected in the stroma fraction of chloroplasts by a proteomic study |
| GO:0009507 chloroplast | HDA PMID:25293756 A proteomic strategy for global analysis of plant protein co... | MODIFY | Summary: High-throughput proteomic detection (Aryal et al. 2014) attributed to chloroplast. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | MODIFY | Summary: AtSubP sequence-based prediction of chloroplast targeting. Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:19597944 all four IPMI subunits of Arabidopsis are predicted to have chloroplast targeting sequences |
| GO:0009536 plastid | HDA PMID:22923678 Putative glycosyltransferases and other plant Golgi apparatu... | MODIFY | Summary: Plastid assignment from LOPIT organelle proteomics (Nikolovski et al. 2012). Reason: Refine the correct general organelle assignment to chloroplast stroma, an already annotated descendant supported by stromal localization/proteomics. The original source fields are preserved. Proposed replacements: chloroplast stroma Supporting Evidence: PMID:22923678 we identified the subcellular localization of 1,110 proteins with high confidence. PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0009570 chloroplast stroma | HDA PMID:18633119 Quantitative proteomics of a chloroplast SRP54 sorting mutan... | ACCEPT | Summary: Detected in chloroplast proteome in a quantitative proteomics study of cpSRP54 mutants (Rutschow et al. 2008). Reason: Stroma localization agrees with IDA data for the IPMI heterodimer (He et al. 2010) and with other stromal proteomes. Supporting Evidence: PMID:18633119 a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0009570 chloroplast stroma | HDA PMID:20061580 AT_CHLORO, a comprehensive chloroplast proteome database wit... | ACCEPT | Summary: AT_CHLORO subplastidial proteomics assigned IIL1 to the stroma. Reason: Consistent with IDA stromal localization of LeuC/LeuD heterodimers (He et al. 2010). Chloroplast stroma is the functional location of the Leu and Met chain-elongation IPMI. Supporting Evidence: PMID:20061580 We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0009570 chloroplast stroma | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping (Plastid, chloroplast stroma). Reason: UniProt location is itself based on experimental evidence (PMID:18431481, PMID:20663849). Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. |
| GO:0016836 hydro-lyase activity | IEA GO_REF:0000002 | MODIFY | Summary: Generic hydro-lyase activity from InterPro family signature. Reason: Refine general hydro-lyase activity to both supported substrate-specific descendant activities of IIL1-containing heterodimers. Neither activity is assigned as autonomous subunit catalysis in the core. Proposed replacements: 3-isopropylmalate dehydratase activity 2-(omega-methylthio)alkylmalate dehydratase activity Supporting Evidence: PMID:19597944 The IPMI substrate, 2-IPM, accumulates when transcript of IPMI LSU1 is reduced sufficiently. |
| GO:0019761 glucosinolate biosynthetic process | IMP PMID:19493961 Omics-based approaches to methionine side chain elongation i... | ACCEPT | Summary: atleuc1 (IIL1) T-DNA knockouts have reduced long-chain (C4-C8) Met-derived glucosinolates with elevated C3 species (Sawada et al. 2009); also shown by Knill et al. 2009. Reason: IIL1 catalyses (as part of IPMI heterodimers with SSU2/SSU3) the isomerization step of each Met chain-elongation cycle, a direct catalytic contribution to aliphatic glucosinolate biosynthesis. Core process. Supporting Evidence: PMID:19493961 In this study we found that knocking out either of the candidate genes, AtleuC1 or AtIMD1 , reduced the level of Met-GSLs, indicating that both genes are actually involved in Met-GSL biosynthesis. PMID:19493961 the levels of Met-GSLs with long chains (C7–C8) were significantly lower in the knockout lines PMID:19597944 The large subunit of Arabidopsis IPMI also participates in the Met chain elongation reactions of aliphatic glucosinolate formation as indicated by the accumulation of SMM in mutant lines PMID:19597944 This metabolite is an oxidation product (enzymatic or non-enzymatic) of the IPMI substrate for the second round of chain elongation |
| GO:0050486 intramolecular hydroxytransferase activity | IMP PMID:19493961 Omics-based approaches to methionine side chain elongation i... | MODIFY | Summary: Intramolecular hydroxytransferase (isomerase class) activity was assigned on the basis of reduced Met-derived glucosinolates in atleuc1 knockouts. Reason: The reaction (2-alkylmalate to 3-alkylmalate via the maleate intermediate) is classified by EC and GO as a hydro-lyase (EC 4.2.1.33 / EC 4.2.1.170), not as an intramolecular hydroxytransferase (EC 5.4.4.-). GO now has a specific term for the Met chain-elongation isomerization, GO:0120528 2-(omega-methylthio)alkylmalate dehydratase activity, which captures what Sawada et al. showed (the gene encodes the MAM-isomerase large subunit). Note Sawada et al. had no in vitro assay; the MF is inferred from mutant metabolite phenotypes. Proposed replacements: 2-(omega-methylthio)alkylmalate dehydratase activity Supporting Evidence: PMID:19493961 In this study we found that knocking out either of the candidate genes, AtleuC1 or AtIMD1 , reduced the level of Met-GSLs, indicating that both genes are actually involved in Met-GSL biosynthesis. PMID:19493961 In addition, a high-throughput in vitro enzyme assay system is required for further confirmation of predicted gene function. PMID:19597944 This metabolite is an oxidation product (enzymatic or non-enzymatic) of the IPMI substrate for the second round of chain elongation |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: 4Fe-4S cluster binding inferred from the aconitase/IPMI large-subunit family signature. Reason: In the aconitase superfamily the [4Fe-4S] cluster is ligated by cysteines in domains 1-3, which reside in the large (LeuC) subunit of heterodimeric IPMIs; UniProt records the cofactor by similarity to E. coli LeuC. Biochemically plausible though not directly demonstrated for the Arabidopsis protein. Supporting Evidence: PMID:19597944 However, in IPMIs of bacteria, archaea and plants domains 1–3 are found in a large subunit, while domain 4 is found in a small subunit. |
| GO:0120528 2-(omega-methylthio)alkylmalate dehydratase activity | EXP PMID:19597944 Arabidopsis thaliana encodes a bacterial-type heterodimeric ... | ACCEPT | Summary: lsu1 mutants accumulate 2-(3'-methylsulfinyl)propylmalate (the oxidized IPMI substrate of the second chain-elongation cycle) and SMM, and show a shift to shorter-chain aliphatic glucosinolates. Reason: Core function for the Met chain-elongation pathway. The activity is exerted by LSU1 paired with SSU2 or SSU3 (the small subunits determine substrate specificity), so at the protein level IIL1 contributes to this activity as part of the heterodimeric complex. No purified-enzyme assay exists; support is from substrate accumulation in vivo. Supporting Evidence: PMID:19597944 This metabolite is an oxidation product (enzymatic or non-enzymatic) of the IPMI substrate for the second round of chain elongation PMID:19597944 The large subunit of Arabidopsis IPMI also participates in the Met chain elongation reactions of aliphatic glucosinolate formation as indicated by the accumulation of SMM in mutant lines PMID:19597944 Thus, the resulting IPMI heterodimer is pathway-specific even if the large subunit per se is not. PMID:32612621 The function of IPMI is defined by the small subunits. |
| GO:0120528 2-(omega-methylthio)alkylmalate dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of EC 4.2.1.170 / associated RHEA reactions (from UniProt curation based on Knill et al. 2009). Reason: Consistent with the experimental EXP annotation for this term. Supporting Evidence: PMID:19597944 This metabolite is an oxidation product (enzymatic or non-enzymatic) of the IPMI substrate for the second round of chain elongation |
| GO:0009316 3-isopropylmalate dehydratase complex | IPI PMID:20663849 Functional specification of Arabidopsis isopropylmalate isom... | NEW | Summary: IIL1 (LeuC) physically interacts with the small subunits (LeuD1-3 = SSU2, SSU3, SSU1) to form functional heterodimeric IPMIs in the chloroplast stroma. Reason: The isomerase activity is a property of the LeuC:LeuD heterodimer (GO:0009316 is defined as the leuC/leuD heterodimer). He et al. 2010 demonstrated the physical interaction and functionality of the heterodimers; Knill et al. 2009 and Lachler et al. 2020 show the small subunit defines pathway specificity. The complex term records the subunit semantics that the 'enables' MF annotations cannot. IIL1 is a structural and catalytic component of the complex, so this is a direct part_of relationship, not a substrate/necessity inference. (Chen et al. 2021 did not detect LeuC-LeuD interaction by Y2H, which is noted as a caveat.) Supporting Evidence: PMID:20663849 We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. PMID:24608865 Isopropylmalate isomerase (IPMI), an enzyme usually involved in Leu biosynthesis, is a heterodimer consisting of a large and a small subunit. PMID:33568694 However, no interactions could be found between LeuC and any of the LeuDs in our Y2H experiments |
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Download this section (compressed HTML)Q: Does IIL1 form stable heterodimers with each of SSU1, SSU2 and SSU3 in planta (He et al. 2010 report interaction, whereas Chen et al. 2021 saw none by Y2H), and what is the relative occupancy of each heterodimer in vascular versus mesophyll plastids?
Q: What are the kinetic parameters of reconstituted LSU1:SSU1, LSU1:SSU2 and LSU1:SSU3 heterodimers toward 2-isopropylmalate and each chain-length 2-(omega-methylthio)alkylmalate?
Q: Is the reported association of IPMI (LeuC) with IPMDH1 part of a functional metabolon that channels methylthioalkylmalates through the chain-elongation cycle?
Experiment: Co-express IIL1 with each small subunit in E. coli leuC/leuD mutants or purify reconstituted heterodimers and measure isomerase kinetics with 2-IPM and 2-(2'-methylthio)ethylmalate / longer homologues.
Type: in vitro enzyme kinetics
Experiment: Native co-immunoprecipitation or affinity purification-MS of tagged IIL1 from leaf chloroplasts to determine which small subunits and other chain-elongation enzymes (e.g. IPMDH1) it associates with in vivo.
Type: protein interaction proteomics
Experiment: Verify [4Fe-4S] cluster content of recombinant IIL1 (EPR/UV-vis) and test oxygen/redox sensitivity of IPMI activity in chloroplast extracts.
Type: biochemistry/spectroscopy
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