MAM3 (METHYLTHIOALKYLMALATE SYNTHASE 3, formerly MAM-L; At5g23020) is a chloroplast-localized 2-oxo-acid/acetyl-CoA condensing enzyme of the isopropylmalate synthase (LeuA-like) family that catalyzes the committed condensation step of the methionine chain-elongation cycle in aliphatic glucosinolate biosynthesis. Unlike MAM1, which handles only the first two cycles, MAM3 accepts all six omega-methylthio-2-oxoalkanoates (C4 to C9 2-oxo acids) used in Arabidopsis and is therefore the principal generator of long-chain (C6-C8) aliphatic glucosinolates and of glucosinolate chain-length diversity; MAM3 loss-of-function mutants lack or are strongly depleted in long-chain glucosinolates, and the defect is complemented by MAM3. MAM3 has broad substrate promiscuity, also condensing 2-oxoisovalerate (isopropylmalate synthase activity), pyruvate (citramalate), phenylpyruvate and the 2-oxo acids of leucine/isoleucine elongation, and can weakly complement an IPMS-deficient E. coli strain, but its IPMS activity is far below that of the dedicated IPMS1/IPMS2 enzymes and it is not thought to contribute materially to leucine biosynthesis. It requires a divalent metal ion and, in contrast to MAM1, is not activated by ATP.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level catalytic activity from InterPro HMGL-like signature; true but uninformative. Reason: The catalytic claim is correct but the supported specific activities already annotated in this review are more informative descendants; refine to these activities. Proposed replacements: methylthioalkylmalate synthase activity Supporting Evidence: PMID:17369439 Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. |
| GO:0003852 2-isopropylmalate synthase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBA transferring IPMS activity from the ancestral IPMS node (PTN000031336). MAM3 retains this ancestral activity at a low level: recombinant MAM3 converts 2-oxoisovalerate to isopropylmalate and complements IPMS-deficient E. coli at 28 C, although its Met-derived 2-oxo acids are strongly preferred. Reason: The IBA is not wrong for MAM3 - there is target-specific experimental evidence that the ancestral activity is retained - but it is a residual promiscuous activity rather than MAM3 core function, and MAM3 is considered unlikely to act as an IPMS in planta. Supporting Evidence: PMID:17369439 MAM3 was able to convert 2-oxoisovalerate (to isopropylmalate) and pyruvate (to citramalate), but based on their kinetic parameters, these were less preferred substrates than the Met-derived 2-oxo acids involved in glucosinolate formation PMID:17369439 Hence, the Arabidopsis MAM3 was able to complement the mutant in the gene encoding IPMS and restored autotrophic growth. PMID:17369439 However, because the specific activity of the IPMSs for 2-oxoisovalerate is at least 40 times higher than those of MAM3, it appears very unlikely that this enzyme is active as IPMS under normal growth conditions |
| GO:0003852 2-isopropylmalate synthase activity | NAS PMID:12432038 Isolation and expression analysis of the isopropylmalate syn... | KEEP AS NON CORE | Summary: Non-traceable author statement from Junk & Mourad (2002), who named the family the IPMS gene family and reported E. coli leuA complementation by the IMS cDNAs; later confirmed by Textor et al. (2007) at low temperature (and not observed by Field et al. 2004). Reason: Weak (NAS) but consistent with later direct assays showing low IPMS activity; retained as a non-core, promiscuous activity. Supporting Evidence: PMID:12432038 cDNAs of IMS2 and IMS3 lacking the 5' chloroplast leader sequence were able to complement a leucine auxotroph of E. coli. PMID:17369439 Previous work showed that MAM3 expression could complement an E. coli mutant ( leuA ) lacking IPMS ( Junk and Mourad, 2002 ). However, Field et al. (2004) failed to observe this complementation. PMID:17369439 MAM3 was able to convert 2-oxoisovalerate (to isopropylmalate) and pyruvate (to citramalate), but based on their kinetic parameters, these were less preferred substrates than the Met-derived 2-oxo acids involved in glucosinolate formation |
| GO:0009098 L-leucine biosynthetic process | TAS PMID:12432038 Isolation and expression analysis of the isopropylmalate syn... | MARK AS OVER ANNOTATED | Summary: Traceable author statement from Junk & Mourad (2002) assigning the IPMS-family gene to leucine biosynthesis. MAM3 has only low IPMS activity (at least 40-fold below IPMS1/IPMS2); its in planta role is glucosinolate chain elongation, and Leu biosynthesis is carried out by IPMS1/IPMS2. Reason: No in planta evidence that MAM3 contributes to Leu biosynthesis; the assignment rests on family membership and heterologous complementation. Not removed outright because a minor contribution cannot be excluded (it has not been tested in an ipms1 ipms2 background). Supporting Evidence: PMID:17369439 However, because the specific activity of the IPMSs for 2-oxoisovalerate is at least 40 times higher than those of MAM3, it appears very unlikely that this enzyme is active as IPMS under normal growth conditions PMID:17369439 However, it is not clear if MAM3 participates in Leu biosynthesis in planta. |
| GO:0009507 chloroplast | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for chloroplast localization; MAM3 own immunolocalization is the descendant evidence, which is expected and not circular. Reason: Consistent with direct immunolocalization. Supporting Evidence: PMID:17369439 Immunolocalization experiments with leaf tissue of Arabidopsis (Col-0) performed with the anti-MAM3 serum confirmed that MAM3 is targeted to the chloroplasts |
| GO:0009507 chloroplast | IDA PMID:17369439 MAM3 catalyzes the formation of all aliphatic glucosinolate ... | ACCEPT | Summary: Immunolocalization with an anti-MAM3 serum (weak cross-reactivity only with MAM1) shows chloroplast targeting in Arabidopsis leaves. Reason: Direct experimental localization; places the MAM condensation step, and thus methionine chain elongation, in the plastid. Supporting Evidence: PMID:17369439 Immunolocalization experiments with leaf tissue of Arabidopsis (Col-0) performed with the anti-MAM3 serum confirmed that MAM3 is targeted to the chloroplasts |
| GO:0009507 chloroplast | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping. Reason: Consistent with experiment. Supporting Evidence: PMID:17369439 Immunolocalization experiments with leaf tissue of Arabidopsis (Col-0) performed with the anti-MAM3 serum confirmed that MAM3 is targeted to the chloroplasts |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | ACCEPT | Summary: AtSubP sequence-based chloroplast prediction. Reason: Consistent with experiment. Supporting Evidence: PMID:17369439 Immunolocalization experiments with leaf tissue of Arabidopsis (Col-0) performed with the anti-MAM3 serum confirmed that MAM3 is targeted to the chloroplasts |
| GO:0010177 methylthioalkylmalate synthase activity | IDA PMID:17369439 MAM3 catalyzes the formation of all aliphatic glucosinolate ... | ACCEPT | Summary: Core molecular function. Purified recombinant MAM3 condenses acetyl-CoA with all six omega-methylthio-2-oxoalkanoates of the Arabidopsis elongation series, with kinetic parameters determined. Reason: Direct enzyme assay, corroborated in vivo by mutant chemotypes and complementation. Supporting Evidence: PMID:17369439 Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. PMID:17369439 Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. |
| GO:0010177 methylthioalkylmalate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from EC 2.3.3.17 / RHEA:50624; matches experimental activity. Reason: Correct. Supporting Evidence: PMID:17369439 Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. |
| GO:0019761 glucosinolate biosynthetic process | IDA PMID:17369439 MAM3 catalyzes the formation of all aliphatic glucosinolate ... | ACCEPT | Summary: MAM3 in vitro activity plus mutant analyses show it produces all glucosinolate chain lengths in vivo. Reason: Core process; MAM3 catalyzes the committed step of each elongation round. Supporting Evidence: PMID:17369439 Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. |
| GO:0019761 glucosinolate biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA electronic annotation; correct. Reason: Consistent with experimental data. Supporting Evidence: PMID:17369439 Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. |
| GO:0019761 glucosinolate biosynthetic process | IMP PMID:15155874 Glucosinolate and amino acid biosynthesis in Arabidopsis. | ACCEPT | Summary: Field et al. (2004): a MAML (MAM3) knockout lacked long-chain aliphatic glucosinolates, restored by complementation; overexpression increased long-chain glucosinolates. Reason: Loss-of-function with complementation; core process. Supporting Evidence: PMID:15155874 A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. |
| GO:0046912 acyltransferase activity, acyl groups converted into alkyl on transfer | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to the parent class of 2-oxo-acid/acyl-CoA condensing enzymes; correct but general. Reason: Refine this correct general acyltransferase term to the experimentally supported descendant activities already reviewed here; MAM3 residual IPMS activity is non-core. Proposed replacements: methylthioalkylmalate synthase activity 2-isopropylmalate synthase activity Supporting Evidence: PMID:17369439 Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. |
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Download this section (compressed HTML)Q: What structural features give MAM3 its broad acceptance of long omega-methylthio-2-oxo acids compared with MAM1, and how does competition between MAM3 and the downstream CYP79F1/F2 for each chain-length intermediate set the final glucosinolate profile?
Q: Does MAM3 contribute measurably to leucine or citramalate formation in planta, e.g. in ipms1 ipms2 backgrounds?
Q: GO has an unused term, GO:0033322 L-homomethionine biosynthetic process, whose definition (formation of L-homomethionine from L-methionine by chain elongation) matches the methionine chain-elongation cycle this enzyme catalyzes. GO obsoleted the related terms GO:0033506 (glucosinolate biosynthetic process from homomethionine; a pathway variant out of scope, replaced by GO:0019761) and GO:0033321 (homomethionine metabolic process; unnecessary grouping), and no gene product in any species carries GO:0033322. Should the chain-elongation enzymes (BCAT4, MAM1/MAM3, IPMI, IPMDH1, BCAT3) be annotated to it, or should GO obsolete it in favour of glucosinolate biosynthetic process?
Experiment: Test whether MAM3 supports Leu synthesis in vivo by combining mam3 with ipms1 ipms2 mutations (or by expressing MAM3 in an ipms1 ipms2 background) and measuring Leu, isopropylmalate and citramalate pools.
Experiment: Engineer MAM3 active-site variants guided by the Brassica juncea MAM structure and express them in mam3 plants to shift glucosinolate chain-length profiles, relevant to tuning glucosinolate content of oilseed Brassicaceae.
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