MAM1 (METHYLTHIOALKYLMALATE SYNTHASE 1, At5g23010; the GSL-ELONG / GSM1 locus) is a plastid-targeted 2-oxo-acid/acetyl-CoA condensing enzyme of the DRE-TIM metallolyase (LeuA-like) family that catalyzes the committed step of each turn of the methionine side-chain elongation cycle in aliphatic glucosinolate biosynthesis: condensation of an omega-methylthio-2-oxoalkanoate with acetyl-CoA to form a 2-(omega-methylthio)alkylmalate. MAM1 evolved from isopropylmalate synthase of leucine biosynthesis by gene duplication and neofunctionalization but has lost IPMS activity; it acts on 4-methylthio-2-oxobutanoate and 5-methylthio-2-oxopentanoate (first and second elongation cycles; trace activity on the C6 acid), requires a divalent metal ion and is activated by ATP. MAM1 therefore determines the predominance of 4-carbon side-chain (C4) aliphatic glucosinolates in Col-0; missense or insertion mutants shift the profile from C4 to C3 glucosinolates. Natural presence/absence and allelic variation at the MAM1/MAM2 locus underlies much of the side-chain-length diversity of glucosinolates among Arabidopsis accessions and Brassica crops. MAM1 expression is co-regulated with BCAT4, is wound-, light- and drought-responsive, and is controlled by the MYB28/MYB29 transcription factors.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level catalytic activity from InterPro HMGL-like/LeuA-type 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:14740211 Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. |
| GO:0003852 2-isopropylmalate synthase activity | IBA GO_REF:0000033 | REMOVE | Summary: PAINT IBA transferring 2-isopropylmalate synthase activity from the ancestral IPMS node (PTN000031336; donors include Arabidopsis IPMS1/IPMS2 and bacterial/yeast LeuA). MAM1 is a neofunctionalized, glucosinolate-specialized descendant: recombinant MAM1 shows no activity with the IPMS substrate 2-oxoisovalerate, is chromatographically separable from plant IPMS, and does not complement an IPMS-null E. coli strain. Reason: Direct MAM1 substrate-specificity assays contradict IPMS activity (PMID:14740211). The ancestral IPMS activity therefore should not propagate through the PAINT node onto this neofunctionalized target. This target-specific challenge is based on biochemical loss of activity, not the number or identity of PAINT donors. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN000031336 SUPPORTS SOURCE BUT NOT TARGET Ancestral IPMS node; the IBD is correct for the bona fide IPMS clade (IPMS1/IPMS2, LeuA) but MAM1 belongs to the neofunctionalized MAM1/MAM2/MAMa lineage that shows no activity with 2-oxoisovalerate and fails to complement IPMS-null E. coli. AGI_LocusCode:AT1G18500 · IPMS1 SUPPORTS SOURCE BUT NOT TARGET Genuine Arabidopsis isopropylmalate synthase of Leu biosynthesis (de Kraker et al. 2007); function not shared by MAM1. AGI_LocusCode:AT1G74040 · IPMS2 SUPPORTS SOURCE BUT NOT TARGET Genuine Arabidopsis isopropylmalate synthase of Leu biosynthesis; function not shared by MAM1. Supporting Evidence: PMID:14740211 However, the MAM1 protein does not show activity with the substrates of any of these other enzymes, and was chromatographically separable from isopropylmalate synthase in extracts of A. thaliana. PMID:15155874 Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. PMID:14740211 Thus, MAM1 is exclusively an enzyme of secondary metabolism, distinct from primary metabolic enzymes catalyzing similar reactions. |
| GO:0009098 L-leucine biosynthetic process | IBA GO_REF:0000033 | REMOVE | Summary: PAINT IBA transferring L-leucine biosynthetic process from the ancestral IPMS node. MAM1 lacks IPMS activity and is exclusively an enzyme of secondary (glucosinolate) metabolism; Arabidopsis Leu biosynthesis is carried out by the bona fide IPMS1 and IPMS2. Reason: Over-propagated process: MAM1 performs no step of leucine biosynthesis. Its condensation chemistry is homologous, but its substrates are the methionine-derived omega-methylthio-2-oxo acids. Propagation Review Root cause: PROPAGATION BAD Failure modes: FUNCTIONAL DIVERGENCE WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN000031336 SUPPORTS SOURCE BUT NOT TARGET Ancestral IPMS node; the IBD is correct for the bona fide IPMS clade (IPMS1/IPMS2, LeuA) but MAM1 belongs to the neofunctionalized MAM1/MAM2/MAMa lineage that acts only on methionine-derived 2-oxo acids and performs no step of Leu biosynthesis. AGI_LocusCode:AT1G18500 · IPMS1 SUPPORTS SOURCE BUT NOT TARGET Genuine Arabidopsis isopropylmalate synthase of Leu biosynthesis (de Kraker et al. 2007); function not shared by MAM1. AGI_LocusCode:AT1G74040 · IPMS2 SUPPORTS SOURCE BUT NOT TARGET Genuine Arabidopsis isopropylmalate synthase of Leu biosynthesis; function not shared by MAM1. Supporting Evidence: PMID:14740211 Thus, MAM1 is exclusively an enzyme of secondary metabolism, distinct from primary metabolic enzymes catalyzing similar reactions. PMID:17189332 These enzymes catalyze the first dedicated step in leucine (Leu) biosynthesis, an aldol-type condensation of acetyl-coenzyme A (CoA) and 2-oxoisovalerate yielding isopropylmalate. PMID:14740211 The A. thaliana MAM1 protein exhibits basic sequence similarity to other previously described enzymes catalyzing the condensation of 2-oxo acids and acetyl-CoA, such as isopropylmalate synthase (EC 2.3.3.13), an enzyme of leucine biosynthesis |
| GO:0009414 response to water deprivation | IEP PMID:23144921 Water stress and aphid feeding differentially influence meta... | KEEP AS NON CORE | Summary: Expression-based: MAM1 transcripts increased about two-fold under drought stress together with MYB29/MYB76 and aliphatic glucosinolate levels. Reason: IEP annotation recording drought-responsive expression of a glucosinolate biosynthetic gene; physiologically informative but not a core function of the enzyme. Supporting Evidence: PMID:23144921 Under drought stress, MAM1 and two transcription factors involved in aliphatic glucosinolate biosynthesis - MYB29 and MYB76 - were up-regulated two-fold |
| GO:0009507 chloroplast | HDA PMID:18431481 Sorting signals, N-terminal modifications and abundance of t... | ACCEPT | Summary: Chloroplast proteome MS identification; consistent with the predicted plastid transit peptide and with the plastid location of the MAM step of methionine chain elongation. Reason: The MAM1 HDA chloroplast annotation and its transit peptide support this location; MAM3 immunolocalization is corroborating paralog evidence, not an assay of MAM1. Supporting Evidence: PMID:18431481 mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins PMID:17369439 confirmed that MAM3 is targeted to the chloroplasts ( Fig. 2 ) as predicted on the basis of its N-terminal sequence ( Kroymann et al., 2001 ) |
| GO:0009507 chloroplast | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA based on the chloroplast localization of MAM3 (AT5G23020, node PTN000814725); MAM1 is the tandem paralog with a comparable transit peptide. Reason: The MAM1 HDA chloroplast annotation and its transit peptide support this location; MAM3 immunolocalization is corroborating paralog evidence, not an assay of MAM1. Supporting Evidence: PMID:17369439 confirmed that MAM3 is targeted to the chloroplasts ( Fig. 2 ) as predicted on the basis of its N-terminal sequence ( Kroymann et al., 2001 ) |
| GO:0009507 chloroplast | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping (Plastid, chloroplast; by similarity). Reason: The MAM1 HDA chloroplast annotation and its transit peptide support this location; MAM3 immunolocalization is corroborating paralog evidence, not an assay of MAM1. Supporting Evidence: PMID:17369439 confirmed that MAM3 is targeted to the chloroplasts ( Fig. 2 ) as predicted on the basis of its N-terminal sequence ( Kroymann et al., 2001 ) |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | ACCEPT | Summary: AtSubP sequence-based chloroplast prediction. Reason: The MAM1 HDA chloroplast annotation and its transit peptide support this location; MAM3 immunolocalization is corroborating paralog evidence, not an assay of MAM1. Supporting Evidence: PMID:17369439 confirmed that MAM3 is targeted to the chloroplasts ( Fig. 2 ) as predicted on the basis of its N-terminal sequence ( Kroymann et al., 2001 ) |
| GO:0009507 chloroplast | ISS PMID:11706188 A gene controlling variation in Arabidopsis glucosinolate co... | ACCEPT | Summary: ISS chloroplast localization from Kroymann et al. (2001), based on the predicted N-terminal plastid transit peptide shared with IPMS homologs. Reason: The MAM1 HDA chloroplast annotation and its transit peptide support this location; MAM3 immunolocalization is corroborating paralog evidence, not an assay of MAM1. Supporting Evidence: PMID:17369439 confirmed that MAM3 is targeted to the chloroplasts ( Fig. 2 ) as predicted on the basis of its N-terminal sequence ( Kroymann et al., 2001 ) |
| GO:0009625 response to insect | IEP PMID:23144921 Water stress and aphid feeding differentially influence meta... | KEEP AS NON CORE | Summary: Expression-based: MAM1 transcript levels decreased after feeding by the aphids Myzus persicae and Brevicoryne brassicae. Reason: IEP; aphid-feeding down-regulation of expression is a response pattern, not a function of the enzyme. The defensive relevance of MAM1 is captured through its role in glucosinolate biosynthesis. Supporting Evidence: PMID:23144921 transcript levels of biosynthetic genes MAM1, CYP79F1, CYP83A1, and UGT74C1 decreased after infestation with either aphid species |
| GO:0010177 methylthioalkylmalate synthase activity | IDA PMID:14740211 Biosynthesis of methionine-derived glucosinolates in Arabido... | ACCEPT | Summary: Core molecular function. Recombinant MAM1 condenses acetyl-CoA with 4-methylthio-2-oxobutanoate and 5-methylthio-2-oxopentanoate (first two elongation cycles), with metal and ATP dependence characterized. Reason: Direct enzyme assay with recombinant protein, corroborated by independent assays (Benderoth et al. 2006) and mutant chemotypes. Supporting Evidence: PMID:14740211 Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. PMID:16754868 Thus, Col-0 MAM1 controls the committed step in both the first and second round of methionine carbon chain elongation, ultimately leading to the formation of aliphatic glucosinolates with four methylene groups (4C) in their side chain. |
| GO:0010177 methylthioalkylmalate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from EC 2.3.3.17 / RHEA:50624 (ARBA and UniProt), matching the experimentally established activity. Reason: Correct. Supporting Evidence: PMID:14740211 Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. |
| GO:0019761 glucosinolate biosynthetic process | IDA PMID:11706188 A gene controlling variation in Arabidopsis glucosinolate co... | ACCEPT | Summary: Kroymann et al. (2001) showed that heterologously expressed MAM1 condenses MTOB with acetyl-CoA (the initial reaction of Met chain elongation), placing MAM1 in aliphatic glucosinolate biosynthesis. Reason: Participation is direct: MAM1 catalyzes the committed step of each of the first two elongation cycles. Abstract-only in cache but the abstract states the assay. Supporting Evidence: PMID:11706188 In cell-free enzyme assays, a heterologously expressed cDNA from this locus was capable of condensing 2-oxo-4-methylthiobutanoic acid with acetyl-coenzyme A, the initial reaction in Met chain elongation. |
| GO:0019761 glucosinolate biosynthetic process | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning electronic annotation to glucosinolate biosynthetic process; correct. Reason: Consistent with experimental data. Supporting Evidence: PMID:14740211 Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. |
| GO:0019761 glucosinolate biosynthetic process | IMP PMID:11706188 A gene controlling variation in Arabidopsis glucosinolate co... | ACCEPT | Summary: Two allelic missense mutants at the GSL-ELONG locus deficient in 4C glucosinolates carry mutations in MAM1, establishing its in vivo role in side-chain elongation. Reason: Loss-of-function genetics plus enzyme activity; core process. Supporting Evidence: PMID:11706188 Two allelic mutants deficient in four-carbon side-chain glucosinolates were shown to contain independent missense mutations within this gene. PMID:17369439 Plant lines with mutations in MAM1 showed a decrease in C 4 glucosinolates and a corresponding increase in C 3 glucosinolates. |
| GO:0019761 glucosinolate biosynthetic process | IMP PMID:16668374 Biochemical Genetics of Plant Secondary Metabolites in Arabi... | ACCEPT | Summary: Haughn et al. (1991) isolated the gsm1 (TU1) mutant lacking or reduced in many aliphatic glucosinolates; gsm1 was later shown to be a MAM1 allele (gsm1-1 = TU1 lacks functional MAM1). Reason: Classic mutant phenotype for the GSM1/MAM1 locus; abstract-only in cache but the allele identity is confirmed by later work. Supporting Evidence: PMID:16668374 Leaf and seed tissues of line TU1 lack or have reduced amounts of many of the aliphatic glucosinolates found in the wild type due to a recessive allele, gsm1, of a single nuclear gene, GSM1. PMID:17369439 Furthermore, MAM activity was detected in protein extracts of a mutant plant line ( gsm 1 - 1 = TU1 ) lacking a functional MAM1 allele |
| GO:0046912 acyltransferase activity, acyl groups converted into alkyl on transfer | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to the parent class of acyl-CoA/2-oxo acid condensing enzymes (acyl groups converted into alkyl on transfer); correct but general. GO:0010177 is its specific child. 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 Supporting Evidence: PMID:14740211 Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. |
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Download this section (compressed HTML)Q: Which N-terminal active-site residues of MAM1 restrict it to the C4/C5 omega-methylthio-2-oxo acids while excluding 2-oxoisovalerate, and can these be engineered to tune glucosinolate chain length in Brassica oilseeds?
Q: How does ATP activation of MAM1 (absent in MAM3) regulate flux into the first elongation cycles in vivo?
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: Solve the structure of Arabidopsis MAM1 with bound substrate and compare with IPMS1/2 and Brassica juncea MAM to map residues responsible for loss of IPMS activity; test by reciprocal mutagenesis and E. coli leuA complementation.
Experiment: Generate mam1 mam3 double mutants by CRISPR (previously unobtainable by crossing) to test whether any aliphatic glucosinolate synthesis persists without MAM activity.
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