Figure 3: CaTHL senses redox environment through a reversible disulfide bond.

(a–c) Activity recovery test of CaTHL, ZrTHL and EcTHL proteins. To investigate how thiolases from different organisms sense redox environment, CaTHL, ZrTHL and EcTHL proteins were treated with various concentrations of H₂O₂ and thiolase activity was measured. DTT with 10 mM final concentration was then added to switch the environment to a reduced state, and compared the activity recovery. Over 80% of activity was recovered from CaTHL that treated with even 500 μM H₂O₂ (a), whereas almost no activity was recovered from ZrTHL and EcTHL that treated with more than 400 μM H₂O₂ (b,c). (d) Activity of four thiolases at various concentrations of DTT. Thiolase activity of CaTHL proteins of wild-type and the CaTHL^{V77Q/N153Y/A286K} mutant, ZrTHL and EcTHL were measured with various concentrations of DTT (0, 1, 5 and 10 mM). While the wild-type CaTHL protein shows only ∼30% activity compared with those of ZrTHL and EcTHL, the activity of the CaTHL^{V77Q/N153Y/A286K} mutant shows more than threefold increase compared with the wile-type enzyme, which were correspondent to similar or even higher activity compared with EcTHL and ZrTHL. Error bars indicate standard deviation among three independently repeated experiments.