Succinyl-CoA:3-sulfinopropionate CoA-transferase from Variovorax paradoxus strain TBEA6, a novel member of the class III coenzyme A (CoA)-transferase family.
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Directly clones, purifies, and characterizes Act(TBEA6) (B9U6P4) from Variovorax paradoxus TBEA6 as a succinyl-CoA:3-sulfinopropionate CoA-transferase of the class III CoA-transferase family.
"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."
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Shows that purified Act(TBEA6) is a 96 +/- 3 kDa homodimer that uses succinyl-CoA (Km = 0.08 mM, Vmax = 44.6 umol min^-1 mg^-1), itaconyl-CoA, or glutaryl-CoA to convert 3-sulfinopropionate (Km = 5.9 mM, Vmax = 46.8 umol min^-1 mg^-1) into 3-sulfinopropanoyl-CoA.
"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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Demonstrates that an unmarked delta-act deletion mutant still grows on TDP and 3SP (indicating a backup 3SP-activating enzyme in V. paradoxus TBEA6), whereas the original Tn5::mob insertion in act blocked 3SP growth via a polar effect on the downstream acd gene (B9U6P5).
"Nonetheless, the V. paradoxus Δact mutant did
not reproduce the phenotype of the Tn5::mob-induced mutant. This defined
deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like
the wild type. Complementation of the Tn5::mob-induced mutant with
pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar
effect of the Tn5::mob transposon on acd(TBEA6), located downstream of
act(TBEA6)."