BCAT4 (BRANCHED-CHAIN AMINOTRANSFERASE 4, At3g19710) is a cytosolic, pyridoxal 5'-phosphate-dependent class-IV aminotransferase that, despite its membership in the branched-chain aminotransferase family, is specialized for methionine. It catalyzes the committed, initial step of methionine side-chain elongation in the biosynthesis of methionine-derived (aliphatic) glucosinolates: transamination of L-methionine to 4-methylthio-2-oxobutanoate (MTOB), which is then condensed with acetyl-CoA by methylthioalkylmalate synthases (MAM1/MAM3) in the plastid. Recombinant BCAT4 is most active with methionine, homomethionine and their 2-oxo acids, converts leucine only at lower affinity, and barely acts on isoleucine and valine; it cannot rescue the branched-chain amino acid auxotrophy of a yeast bat1/bat2 mutant. bcat4 knockouts have about half the wild-type level of aliphatic glucosinolates and accumulate free methionine and S-methylmethionine. BCAT4 is expressed mainly in phloem, is transiently induced by wounding and follows a light-dependent diurnal pattern co-ordinated with MAM1. Its function is partially backed up by the plastid BCAT3.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Generic catalytic activity inferred from the class-IV aminotransferase InterPro signatures. Correct but uninformative; the specific activity is L-methionine:oxo-acid transaminase activity (GO:0010326), which is annotated with direct experimental evidence. 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: L-methionine:oxo-acid transaminase activity Supporting Evidence: PMID:17056707 Recombinant BCAT4 showed high efficiency with Met and its derivatives and the corresponding 2-oxo acids |
| GO:0004084 branched-chain-amino-acid:2-oxoglutarate transaminase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference of the ancestral branched-chain aminotransferase activity of the plant BCAT clade (seeded by BCAT1, BCAT2, BCAT3, BCAT6). BCAT4 descends from that node and retains measurable in vitro activity with leucine (and weak activity with isoleucine), but its substrate preference has shifted to methionine and its derivatives, and it cannot support BCAA synthesis in yeast. Reason: The IBD node placement is sound for the family and BCAT4 is inside the clade; the activity is not lost outright (Leu is converted at about 5-fold lower affinity than Met, and Funakoshi et al. reported activity with Leu, Ile and Val as donors with 2-oxoglutarate). However, the physiologically relevant activity is methionine transamination, and BCAT4 fails to complement a yeast BCAA-auxotroph, so BCAA:2-oxoglutarate transamination is a residual, non-core activity rather than the gene's function. Supporting Evidence: PMID:17056707 Substantial activities were also measured with homomethionine (37% at 1 mM and 65% at 5 mM) and with Leu (29% at 1 mM and 62% at 5 mM), while only little or no activity was detected with Ile (8% at 5 mM) and Val PMID:17056707 Thus, the affinity of BCAT4 to Leu is about five times lower than to Met. PMID:12068099 No complementation was achieved with AtBCAT-4 (data not shown). |
| GO:0004084 branched-chain-amino-acid:2-oxoglutarate transaminase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the branched-chain aminotransferase family signatures. As for the IBA row, the activity is residual for BCAT4, whose preferred substrates are methionine and its chain-elongated homologs. Reason: Family-level signature mapping cannot distinguish the methionine-specialized BCAT4 from the canonical BCAA transaminases. The activity is measurable in vitro (Leu) but is not BCAT4's physiological role. Supporting Evidence: PMID:17056707 The standard substrates for BCATs are, with the exception of Leu and its 2-oxo acid, only poorly converted if at all. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | MODIFY | Summary: Correct but general. BCAT4 is a soluble cytosolic enzyme (GFP fusion and fractionation); cytosol (GO:0005829) is the more precise location. Reason: Refine cytoplasm to the experimentally supported cytosol descendant already annotated; GFP and fractionation independently locate BCAT4 there. Proposed replacements: cytosol Supporting Evidence: PMID:17056707 green fluorescent protein tagging experiments and subcellular protein fractions show that BCAT4 is located in the cytosol |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | MODIFY | Summary: AtSubP sequence-based prediction of cytoplasmic localization; agrees with the absence of organellar targeting signals and with experimental data. Reason: Refine cytoplasm to the experimentally supported cytosol descendant already annotated; GFP and fractionation independently locate BCAT4 there. Proposed replacements: cytosol Supporting Evidence: PMID:12068099 For AtBCAT-4 and -6, targeting to plastids or mitochondria is clearly excluded |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of cytosolic activity for the BCAT4/BCAT6 subclade; BCAT4's own experimental cytosol annotation is among the descendant evidence, which is expected and not circular. Reason: Matches direct experimental localization of BCAT4 to the cytosol. Supporting Evidence: PMID:17056707 Taken together, the two independent experimental approaches consistently show that BCAT4 is a cytosolic enzyme and is thus located in a compartment different from the MAM enzymes. |
| GO:0005829 cytosol | IDA PMID:17056707 Branched-chain aminotransferase4 is part of the chain elonga... | ACCEPT | Summary: GFP fusion in tobacco protoplasts gave exclusively cytosolic fluorescence, and an affinity-purified antibody detected BCAT4 only in the cytosolic fraction, not in chloroplast or mitochondrial fractions. Reason: Two independent direct assays; the cytosolic location of the first step of methionine chain elongation is a notable feature of the pathway (the following MAM step is plastidial). Supporting Evidence: PMID:17056707 The green fluorescence is seen exclusively in the cytosol ( Figure 5A ). PMID:17056707 considering the strong enrichment of the cytosol and chloroplast fractions and the exclusive detection of BCAT4 in the cytosol, these results clearly show that BCAT4 is a cytosolic protein. |
| GO:0009081 branched-chain amino acid metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Family-level InterPro2GO mapping. BCAT4 has not been shown to function in leucine/isoleucine/valine metabolism in vivo: it fails to complement BCAA auxotrophy, and bcat4 knockouts change methionine/S-methylmethionine and aliphatic glucosinolate levels rather than BCAA pools. Reason: The family mapping over-reaches for this neofunctionalized, methionine-specific member. The in vivo metabolic role of BCAT4 is methionine chain elongation for glucosinolate biosynthesis, not BCAA metabolism. Not removed outright because recombinant BCAT4 does convert leucine in vitro. Supporting Evidence: PMID:17056707 In a previous complementation analysis, BCAT4 failed to rescue the auxotrophy of a yeast double knockout mutant for BCAAs, suggesting a function other than typical BCAA metabolism PMID:17056707 All other proteinogenic amino acids measured in this tissue did not show any clear changes, except Thr, which showed an increase of βΌ25%. |
| GO:0009416 response to light stimulus | IEP PMID:17056707 Branched-chain aminotransferase4 is part of the chain elonga... | KEEP AS NON CORE | Summary: BCAT4 transcript levels rise up to fivefold on illumination and stay high in the light, in phase with MAM1. This is an expression pattern, not a role of the enzyme in light perception or signalling. Reason: IEP expression-pattern annotation; valid as a record of light-regulated expression but not part of BCAT4's core function. Supporting Evidence: PMID:17056707 While only low BCAT4 mRNA levels were observed during the night, an up to fivefold increase was found upon illumination ( Figure 4 , top panel). |
| GO:0009611 response to wounding | IEP PMID:17056707 Branched-chain aminotransferase4 is part of the chain elonga... | KEEP AS NON CORE | Summary: BCAT4 promoter activity and mRNA are transiently induced by mechanical wounding (about 2-fold within 5 min), consistent with inducible glucosinolate defense. Reason: IEP; documents wound-inducible transcription of a defense-metabolite biosynthetic gene, which is physiologically meaningful but not a core activity of the enzyme. Supporting Evidence: PMID:17056707 A 1.9-fold increase of BCAT4 steady state mRNA level can already be observed 5 min after wounding |
| GO:0010326 L-methionine:oxo-acid transaminase activity | EXP PMID:18318836 Cloning and functional characterization of Arabidopsis thali... | ACCEPT | Summary: Funakoshi et al. (2008) cloned the Arabidopsis D-amino acid aminotransferase and, per UniProt, also characterized BCAT4 kinetically (Km 1.61 mM for L-leucine; no activity with D-isomers). Only the abstract is cached, which does not mention BCAT4, but the activity is independently well established for BCAT4. Reason: The cached record is abstract-only and centres on a different enzyme, so the exact assay cannot be checked here; per project policy we defer to the curator because the annotated function is clearly correct for BCAT4 (Schuster et al. 2006 directly demonstrate methionine transamination). Supporting Evidence: PMID:17056707 For Met, K m and V max values were calculated to be 0.93 Β± 0.08 mM and 0.089 Β± 0.002 ΞΌmol/minΒ·mg protein, respectively. PMID:17056707 This sharp increase of free Met and its transport derivative SMM further support the conclusion that BCAT4 catalyzes the initial transamination of Met in glucosinolate formation generating MTOB. |
| GO:0010326 L-methionine:oxo-acid transaminase activity | IDA PMID:17056707 Branched-chain aminotransferase4 is part of the chain elonga... | ACCEPT | Summary: Core molecular function. Recombinant BCAT4 transaminates L-methionine (Km 0.93 mM) and its 2-oxo acid MTOB (Km 0.045 mM) with Michaelis-Menten kinetics, and prefers methionine over all other amino acids tested. Reason: Direct enzyme assay with purified recombinant protein, supported in vivo by methionine and S-methylmethionine accumulation in two independent knockout alleles. Supporting Evidence: PMID:17056707 For Met, K m and V max values were calculated to be 0.93 Β± 0.08 mM and 0.089 Β± 0.002 ΞΌmol/minΒ·mg protein, respectively. PMID:17056707 This sharp increase of free Met and its transport derivative SMM further support the conclusion that BCAT4 catalyzes the initial transamination of Met in glucosinolate formation generating MTOB. |
| GO:0010326 L-methionine:oxo-acid transaminase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic mapping from the UniProt EC 2.6.1.88 / RHEA:31763 catalytic-activity annotation, which itself rests on the experimental data for BCAT4. Reason: Correct, and matches the experimentally supported core activity. Supporting Evidence: PMID:17056707 strongly implicate BCAT4 in catalysis of the initial deamination of Met to 4-methylthio-2-oxobutyrate |
| GO:0019761 glucosinolate biosynthetic process | IMP PMID:17056707 Branched-chain aminotransferase4 is part of the chain elonga... | ACCEPT | Summary: Two independent T-DNA knockouts (bcat4-1, bcat4-2; no detectable protein) reduce methionine-derived glucosinolates to about 50% of wild type in leaves and seeds, while indolic glucosinolates are not reduced. BCAT4 catalyzes the entry step of methionine chain elongation, so it directly participates in aliphatic glucosinolate biosynthesis. Reason: Core biological process: BCAT4 performs a catalytic step (Met deamination) of the pathway, satisfying participation; loss-of-function phenotype confirms the in vivo role. Supporting Evidence: PMID:17056707 This was substantiated by in vivo analysis of two BCAT4 T-DNA knockout mutants, in which Met-derived aliphatic glucosinolate accumulation is reduced by approximately 50%. PMID:17056707 By contrast, total indolic glucosinolates were slightly increased in the mutants. |
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Download this section (compressed HTML)Q: Does BCAT4 also catalyze the transamination of chain-elongated 2-oxo acids back to homomethionine/dihomomethionine in the cytosol, or is that restricted to plastid BCAT3?
Q: How is MTOB produced in the cytosol by BCAT4 transported into the chloroplast for MAM condensation (candidate transporter BAT5/BASS5)?
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: Determine kinetic parameters of recombinant BCAT4 with the full series of omega-methylthio-2-oxoalkanoates (C5-C9) and 2-oxoglutarate versus other amino acceptors to define which transamination steps of the elongation cycle it can serve.
Experiment: Use 13C/15N-labelled methionine feeding in bcat4, bcat3 and bcat3 bcat4 plants to quantify flux through the cytosolic versus plastidial entry into methionine chain elongation.
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