Act (Act(TBEA6), VPARA_05450, KLN58430.1) is a 398-amino-acid cytosolic homodimeric (96 +/- 3 kDa) succinyl-CoA:3-sulfinopropionate CoA-transferase (EC 2.8.3.-; family III CoA-transferase Pfam PF02515, InterPro IPR003673) characterized in Variovorax paradoxus strain TBEA6. Encoded immediately upstream of the 3-sulfinopropanoyl-CoA desulfinase gene acd (B9U6P5, VPARA_05440), Act catalyzes the reversible transfer of CoA from succinyl-CoA (apparent Vmax = 44.6 umol min^-1 mg^-1, Km = 0.08 mM; kcat = 36.0 s^-1), itaconyl-CoA, or glutaryl-CoA to 3-sulfinopropionate (3SP; apparent Vmax = 46.8 umol min^-1 mg^-1, Km = 5.9 mM; kcat = 37.7 s^-1) to form 3-sulfinopropanoyl-CoA (3SP-CoA) during the catabolism of 3,3'-thiodipropionate (TDP) and 3-sulfinopropionate.
Definition: Catalysis of the reaction: succinyl-CoA + 3-sulfinopropanoate = succinate + 3-sulfinopropanoyl-CoA (EC 2.8.3.-).
Justification: Purified and kinetically characterized in Variovorax paradoxus strain TBEA6 Act(TBEA6) (UniProtKB:B9U6P4; PMID:23772073), where it catalyzes the physiological activation of 3-sulfinopropionate to 3-sulfinopropanoyl-CoA during 3,3'-thiodipropionate degradation, but lacks a specific child term under GO:0008410 (CoA-transferase activity).
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level catalytic activity term; refine to CoA-transferase activity (GO:0008410). Reason: GO:0003824 (catalytic activity) is overly general for a biochemically characterized class III succinyl-CoA:3-sulfinopropionate CoA-transferase (PMID:23772073); refining to GO:0008410 (CoA-transferase activity), which is already present on this record, captures its specific family III CoA-transferase mechanism. Proposed replacements: CoA-transferase activity Supporting Evidence: PMID:23772073 The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. |
| GO:0008410 CoA-transferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Directly verified experimentally in PMID:23772073: Act(TBEA6) (B9U6P4) is a homodimeric class III succinyl-CoA:3-sulfinopropionate CoA-transferase. Reason: Purified recombinant V. paradoxus TBEA6 Act (B9U6P4) catalyzes CoA transfer from succinyl-CoA (Vmax = 44.6 umol min^-1 mg^-1, Km = 0.08 mM), itaconyl-CoA, or glutaryl-CoA to 3-sulfinopropionate (Vmax = 46.8 umol min^-1 mg^-1, Km = 5.9 mM) to yield 3-sulfinopropanoyl-CoA (PMID:23772073). Because GO currently lacks a specific child term for succinyl-CoA:3-sulfinopropionate CoA-transferase activity, GO:0008410 is the most specific valid GO Molecular Function term. Supporting Evidence: PMID:23772073 Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 μmol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 μmol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. |
| GO:0033877 succinyl-CoA:(R)-benzylsuccinate CoA-transferase activity | IEA GO_REF:0000003 | REMOVE | Summary: Spurious EC 2.8.3.15 mapping from preliminary WGS annotation; Act(TBEA6) is a homodimeric succinyl-CoA:3-sulfinopropionate CoA-transferase, not a heterotetrameric BbsEF benzylsuccinate CoA-transferase. Reason: GO:0033877 derives from an automated EC 2.8.3.15 assignment on the WGS submission EMBL:KLN58430.1. Direct biochemical characterization of B9U6P4 (Act(TBEA6)) in PMID:23772073 demonstrated that it is a homodimeric succinyl-CoA:3-sulfinopropionate CoA-transferase (EC 2.8.3.x) involved in aerobic 3,3'-thiodipropionate catabolism, not the anaerobic heterotetrameric BbsE/BbsF succinyl-CoA:(R)-benzylsuccinate CoA-transferase (EC 2.8.3.15) of toluene degradation. Supporting Evidence: PMID:23772073 The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. PMID:23772073 Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 μmol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 μmol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. |
| GO:0044273 sulfur compound catabolic process | IDA PMID:23772073 Succinyl-CoA:3-sulfinopropionate CoA-transferase from Variov... | NEW | Summary: Act(TBEA6) activates 3-sulfinopropionate to 3-sulfinopropanoyl-CoA in the 3,3'-thiodipropionate (TDP) and 3-sulfinopropionate (3SP) organosulfur catabolic pathway. Reason: Biochemical characterization of purified Act(TBEA6) in V. paradoxus TBEA6 (PMID:23772073) established that it directly catalyzes the CoA-activation of 3-sulfinopropionate (3SP) to 3-sulfinopropanoyl-CoA (3SP-CoA) upstream of Acd-mediated desulfination (PMID:24317404) during catabolism of the organosulfur compounds TDP and 3SP. An unmarked delta-act deletion mutant still grows on TDP and 3SP like wild type due to functional redundancy from an uncharacterized backup CoA-activating enzyme, whereas the earlier Tn5::mob insertion in act (PMID:19001372) caused 3SP accumulation via a polar effect on downstream acd (PMID:23772073). Supporting Evidence: PMID:23772073 The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act(TBEA6) (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3'-thiodipropionate (TDP) degradation. |
| GO:0005829 cytosol | IC UniProtKB:B9U6P4 | NEW | Summary: Act(TBEA6) is a soluble cytosolic homodimeric CoA-transferase lacking signal peptides or transmembrane helices. Reason: Inferred by curator (IC) because Act(TBEA6) (B9U6P4) is a 398-residue soluble family III CoA-transferase lacking signal peptides or transmembrane helices (UniProtKB:B9U6P4) that acts on intracellular 3-sulfinopropionate and succinyl-CoA pools to supply 3-sulfinopropanoyl-CoA to cytosolic Acd (note that analytical size-exclusion chromatography of the 96 +/- 3 kDa homodimer in PMID:23772073 was performed on recombinant His-tagged protein purified from E. coli). Supporting Evidence: PMID:23772073 Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V(max) of 44.6 μmol min(-1) mg(-1) for succinyl-CoA, which corresponds to a turnover number of 36.0 s(-1) per subunit of Act(TBEA6). For 3SP, the apparent V(max) was determined as 46.8 μmol min(-1) mg(-1), which corresponds to a turnover number of 37.7 s(-1) per subunit of Act(TBEA6). The apparent K(m) values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. |
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Download this section (compressed HTML)Q: Which paralogous acyl-CoA transferase or acyl-CoA synthetase in Variovorax paradoxus TBEA6 compensates for the loss of act in the unmarked delta-act deletion mutant during growth on 3,3'-thiodipropionate and 3-sulfinopropionate?
Experiment: Perform comparative transcriptomics and activity-guided fractionation of cell lysates from the Variovorax paradoxus TBEA6 delta-act deletion mutant grown on 3-sulfinopropionate vs. succinate.
Hypothesis: At least one alternative CoA-transferase or ATP-dependent acyl-CoA synthetase is induced during growth on 3SP and catalyzes backup 3SP-CoA formation in the delta-act background.
Type: RNA-seq and activity-guided protein purification in delta-act mutant
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