FMO GS-OX1 (At1g65860) is a FAD- and NADPH-dependent flavin-containing monooxygenase of the crucifer-specific FMO GS-OX subclade (clade III plant FMOs) that catalyzes the S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates in methionine-derived (aliphatic) glucosinolate biosynthesis. This side-chain modification converts, for example, 4-methylthiobutyl glucosinolate (glucoerucin) into 4-methylsulfinylbutyl glucosinolate (glucoraphanin), the precursor of the isothiocyanate sulforaphane, and supplies the methylsulfinylalkyl substrates that 2-oxoglutarate-dependent dioxygenases (AOP2/AOP3) further convert to alkenyl or hydroxyalkyl glucosinolates. The enzyme acts on both desulfo- and intact glucosinolates independent of side-chain length (C3 to C8) and requires the S-glucosyl moiety, as it does not oxygenate methionine, chain-elongated methionines or the aldoxime intermediate. It is expressed mainly in leaves and acts redundantly with the paralogs FMO GS-OX2 to GS-OX5; overexpression drives almost complete conversion of methylthioalkyl to methylsulfinylalkyl glucosinolates.
Definition: Catalysis of NADPH- and oxygen-dependent S-oxygenation of an omega-(methylsulfanyl)alkyl glucosinolate to the corresponding omega-(methylsulfinyl)alkyl glucosinolate.
Justification: The substrate-class reaction is EC 1.14.13.237 and RHEA:42208. Current GO chain-specific entries GO:0080103βGO:0080107 have chemically inconsistent propyl wording for longer chains; preserve those sourced annotations, but use this explicitly defined proposed activity for the broad-specificity core. The enzyme also accepts corresponding desulfoglucosinolates.
Parent term: monooxygenase activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004497 monooxygenase activity | IBA GO_REF:0000033 | MODIFY | Summary: FMO GS-OX1 S-oxygenates methylthioalkyl glucosinolates over a range of chain lengths. Reason: Use the proposed substrate-class S-oxygenase activity, defined by the measured methylthioalkyl-to-methylsulfinylalkyl conversion and covering the demonstrated chain-length range. This refines broad monooxygenase activity; replacement of N,N-dimethylaniline monooxygenase is a lateral substrate/reaction correction. The original chain-specific GOA rows are preserved, including their current GO wording, without using chemically inconsistent longer-chain labels as the authored core activity. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN009058586 UNRESOLVED Proximate PAINT node traced from the original annotation. Its full ancestral assertion history has not been reconstructed in this audit; the target-specific assessment is grounded in the primary evidence explained in review.reason, not donor counts or self-inclusion. Proposed replacements: methylthioalkyl glucosinolate S-oxygenase activity |
| GO:0004499 N,N-dimethylaniline monooxygenase activity | IEA GO_REF:0000002 | MODIFY | Summary: FMO GS-OX1 S-oxygenates methylthioalkyl glucosinolates over a range of chain lengths. Reason: Use the proposed substrate-class S-oxygenase activity, defined by the measured methylthioalkyl-to-methylsulfinylalkyl conversion and covering the demonstrated chain-length range. This refines broad monooxygenase activity; replacement of N,N-dimethylaniline monooxygenase is a lateral substrate/reaction correction. The original chain-specific GOA rows are preserved, including their current GO wording, without using chemically inconsistent longer-chain labels as the authored core activity. Proposed replacements: methylthioalkyl glucosinolate S-oxygenase activity |
| GO:0005634 nucleus | ISM GO_REF:0000122 | UNDECIDED | Summary: The predicted nuclear location lacks target-specific resolution. Reason: The biosynthetic role and absence of an annotated nuclear localization signal do not exclude nuclear access by a soluble enzyme. No direct target-localization result resolving this particular prediction was found, so an asserted contradiction is not justified. |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000002 | ACCEPT | Summary: FAD binding inferred from flavin monooxygenase InterPro signatures; FMOs carry a tightly bound FAD prosthetic group (UniProt cofactor: FAD, by similarity). Reason: Correct cofactor-binding annotation consistent with membership of the FMO family and the flavin-dependent S-oxygenation chemistry. Not a core function in itself but an accurate supporting molecular feature. Supporting Evidence: PMID:18799661 It is reported that when there is no available substrate, the FMO proteins exist as 4 Ξ± -hydroperoxy flavin |
| GO:0050661 NADP binding | IEA GO_REF:0000002 | ACCEPT | Summary: NADP(H) binding inferred from FMO InterPro signatures; NADPH is the electron donor for the FMO catalytic cycle (UniProt reaction with NADPH/NADP+, RHEA:42208). Reason: Correct and consistent with the FMO catalytic mechanism in which NADPH reduces the FAD to form the C4a-hydroperoxyflavin oxygenating species. Supporting feature, not core function. Supporting Evidence: PMID:18799661 which is a precharged complex containing a reduced form of fatty acid dehydrogenase and NADPH that is ready to act on substrates |
| GO:0080102 3-methylthiopropyl glucosinolate S-oxygenase activity | IMP PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: S-oxygenation of the C3 methylthioalkyl glucosinolate (3-methylthiopropyl glucosinolate) to 3-methylsulfinylpropyl glucosinolate, inferred from glucosinolate profiles of FMO GS-OX1 knockout/overexpression lines. Reason: FMO GS-OX1 S-oxygenates methylthioalkyl glucosinolates independent of chain length, and overexpression lines show almost complete conversion of methylthioalkyl to methylsulfinylalkyl glucosinolates. The full text (Hansen et al. 2007) is not cached, so the chain-length-specific IMP details cannot be verified directly, but the activity is fully consistent with the biochemistry and we defer to the curator. Supporting Evidence: PMID:17461789 The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs PMID:18799661 FMO GS-OX2 to FMO GS-OX4 are able to catalyze the S -oxygenation independent of chain length, as was observed for FMO GS-OX1 |
| GO:0080103 4-methylthiopropyl glucosinolate S-oxygenase activity | IDA PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: Recombinant FMO GS-OX1 converts 4-methylthiobutyl (4-MTB) glucosinolate (desulfo and intact) to 4-methylsulfinylbutyl (glucoraphanin) glucosinolate. Note that the GO term label reads "4-methylthiopropyl" but the term represents the C4 (methylthiobutyl) S-oxygenation. Reason: Direct biochemical evidence from recombinant protein (Hansen et al. 2007), independently reproduced in Li et al. 2008 with E. coli-expressed FMO GS-OX1. This is the best-characterized reaction of the enzyme and the core molecular function. Supporting Evidence: PMID:17461789 This is evidenced by biochemical characterization of the recombinant protein, and analyses of the GSL content in FMO(GS-OX1) overexpression lines and an FMO(GS-OX1) knock-out mutant of Arabidopsis. PMID:18799661 Consistent with the previous work, FMO GS-OX1 , FMO GS-OX2 , FMO GS-OX3 , and FMO GS-OX4 , but not FMO GS-OX5 , catalyzed the conversion of 4-MTB to 4-MSB |
| GO:0080103 4-methylthiopropyl glucosinolate S-oxygenase activity | IMP PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: In planta support for 4-methylthiobutyl glucosinolate S-oxygenation: knockout plants accumulate methylthiobutyl glucosinolate and overexpressors show ~5-fold increased 4-methylsulfinylbutyl glucosinolate in seeds. Reason: Consistent genetic evidence matching the biochemical activity; core function. Supporting Evidence: PMID:17461789 The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs, with an approximately fivefold increase in 4-methylsulfinylbutyl GSL in seeds. |
| GO:0080104 5-methylthiopropyl glucosinolate S-oxygenase activity | IMP PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: S-oxygenation of 5-methylthiopentyl glucosinolate inferred from glucosinolate profiles of mutant/overexpression lines (UniProt: knockout accumulates methylthiobutyl, -pentyl and -heptyl glucosinolates in leaves). Reason: Consistent with the chain-length-independent S-oxygenase activity of FMO GS-OX1. The full text is not cached; we defer to the curator's reading of the in planta profiles. Supporting Evidence: PMID:17461789 The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs PMID:18799661 FMO GS-OX2 , FMO GS-OX3 , and FMO GS-OX4 S -oxygenated all available MT GSLs as did FMO GS-OX1 |
| GO:0080105 6-methylthiopropyl glucosinolate S-oxygenase activity | IMP PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: S-oxygenation of 6-methylthiohexyl glucosinolate inferred from in planta glucosinolate profiles. Reason: Consistent with the broad chain-length specificity of FMO GS-OX1; full text not cached, curator deferred to. Supporting Evidence: PMID:17461789 The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs PMID:18799661 FMO GS-OX2 , FMO GS-OX3 , and FMO GS-OX4 S -oxygenated all available MT GSLs as did FMO GS-OX1 |
| GO:0080106 7-methylthiopropyl glucosinolate S-oxygenase activity | IMP PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: S-oxygenation of 7-methylthioheptyl glucosinolate inferred from in planta glucosinolate profiles (knockout accumulates methylthioheptyl glucosinolate in leaves per UniProt). Reason: Consistent with the broad chain-length specificity of FMO GS-OX1; full text not cached, curator deferred to. Supporting Evidence: PMID:17461789 The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs |
| GO:0080107 8-methylthiopropyl glucosinolate S-oxygenase activity | IDA PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: Recombinant FMO GS-OX1 S-oxygenates the long-chain 8-methylthiooctyl (8-MTO) glucosinolate to 8-methylsulfinyloctyl glucosinolate. Reason: Directly confirmed with recombinant FMO GS-OX1 incubated with seed-derived desulfo glucosinolates (Li et al. 2008), where all five FMO GS-OX enzymes converted 8-MTO to 8-MSO. Supporting Evidence: PMID:18799661 Interestingly, all five recombinant proteins converted 8-MTO to 8-MSO PMID:18799661 Thus, FMO GS-OX1 , FMO GS-OX2 , FMO GS-OX3 , and FMO GS-OX4 catalyze the S -oxygenating reaction for both short-chain and long-chain MT GSL |
| GO:0080107 8-methylthiopropyl glucosinolate S-oxygenase activity | IMP PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | ACCEPT | Summary: In planta evidence that FMO GS-OX1 contributes to 8-methylsulfinyloctyl glucosinolate production. Reason: Li et al. 2008 explicitly state that knockout analysis shows both FMO GS-OX1 and FMO GS-OX2 control 8-MSO production in planta, supporting this IMP annotation. Supporting Evidence: PMID:18799661 evidenced by the knockout mutant analysis where both FMO GS-OX1 and FMO GS-OX2 control 8-MSO production in planta |
| GO:0019761 glucosinolate biosynthetic process | IDA PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenatin... | NEW | Summary: FMO GS-OX1 catalyzes a step of aliphatic glucosinolate biosynthesis (side-chain S-oxygenation), but no biological process term is currently annotated in GOA. Reason: Participation test: FMO GS-OX1 itself catalyzes the conversion of methylthioalkyl to methylsulfinylalkyl glucosinolates, the first side-chain modification step of the pathway, so it performs part of the work of glucosinolate biosynthesis rather than merely being required for it. Comparator check: other enzymes catalyzing individual steps of aliphatic glucosinolate biosynthesis, e.g. the downstream side-chain-modifying dioxygenase AOP2 (Q945B5, IDA GO:0019761 from PMID:11251105), carry this term, and the process term is not an ancestor/descendant of the molecular function terms already present. The more specific child terms do not cover S-oxygenated aliphatic glucosinolates, so the general term is appropriate. Supporting Evidence: PMID:17461789 Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis. PMID:18799661 This suggests that this cluster of FMOs is involved in GSL biosynthesis and that the enzymes have evolved different substrate specificities. |
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Download this section (compressed HTML)Q: Should GO:0080103βGO:0080107 replace the current longer-chain methylthiopropyl wording with methylthiobutyl, -pentyl, -hexyl, -heptyl and -octyl, and gain a substrate-class parent corresponding to EC 1.14.13.237/RHEA:42208? The current review proposes that parent activity and preserves all original sourced terms.
Q: Where in the cell does FMO GS-OX1 act (cytosol or ER surface), and does it physically associate with the ER-anchored cytochrome P450s of the glucosinolate core pathway?
Q: How is the S-oxygenation workload partitioned in planta among the redundant FMO GS-OX1-5 paralogs across tissues and developmental stages?
Experiment: Determine subcellular localization of FMO GS-OX1 using a functional fluorescent-protein fusion expressed from its native promoter, co-imaged with ER and cytosolic markers.
Experiment: Generate higher-order fmo gs-ox1/2/3/4/5 mutants (e.g. by CRISPR) to test whether S-oxygenation of methylthioalkyl glucosinolates is fully abolished and to assign each paralog's in planta contribution by chain length and tissue.
Experiment: Obtain steady-state kinetic parameters for purified FMO GS-OX1 across C3-C8 methylthioalkyl glucosinolates and solve its structure to define the S-glucosyl recognition determinants.
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