Figure 1.
The reaction catalyzed by MCR.
Figure 2.
Expression of the recombinant MCR protein and its derivatives.
(A) Schematic representation of MCR, MCR-N, and MCR-C proteins fused to a His6-tag (black). (B) Western blot analysis of MCR, MCR-N, and MCR-C recombinant proteins expressed in E. coli BL21(DE3) strain. The soluble protein samples were applied onto SDS-PAGE and detected using anti-His6 antibody.
Figure 3.
Functional domain analysis of MCR protein.
(A) SDS-PAGE of purified MCR, MCR-N, and MCR-C proteins. (B) In vitro analysis of malonyl-CoA reductase activity. Enzyme activities were assayed as described in Materials and Methods. The reaction products of MCR, MCR-N, MCR-C, and MCR-N/MCR-C were analyzed using LC-MS. Representative results from duplicated independent experiments are shown, TIC, total ion chromatogram. MCA, malonyl-CoA. MSA, malonate semialdehyde.
Figure 4.
Kinetic analysis of MCR and MCR-C proteins.
(A) Enzyme activity comparison of MCR, MCR-N/MCR-C, and MCR-C. 0.05 nmol (each) of MCR, MCR-N/MCR-C, and MCR-C were used to catalyze the reactions at pH7.8 at 57°C, respectively. Date represent mean ± standard deviation (N=3). (B) Effects of temperature on enzymatic activity of His6-tagged MCR and MCR-C proteins. The reactions were performed at the condition of pH 7.8 (Tris-HCl). For MCR, 100% corresponds to the enzyme activity of 0.47 mmol/min/μmol protein at 57°C; for MCR-C, 100% corresponds to the enzyme activity of 0.99 mmol/min/μmol protein at 57°C. (C) Effects of pH on enzymatic activity of His6-tagged MCR and MCR-C proteins. The reactions were performed at 57°C. For MCR, 100% corresponds to the enzyme activity of 0.47 mmol/min/μmol protein at the condition of pH7.8 (Tris-HCl); for MCR-C, 100% corresponds to the enzyme activity of 1.12 mmol/min/μmol protein at the condition of pH7.2 (Tris-HCl).
Figure 5.
Site-directed mutagenic analysis of the MCR-N and MCR-C proteins.
(A) The location of amino acid changed in the MCR-N and MCR-C proteins that were tested. (B) In vitro analysis of malonyl-CoA reductase activity. Wild-type and mutated proteins were tested and the products were identified by LC-MS. In MCR-N reactions, wild-type MCR-C protein was used to convert malonyl-CoA into malonate semialdehyde, which is the substrate of MCR-N.
Figure 6.
In vivo effect of separated MCR protein on protein expression (A), enzyme activity (B), and 3HP production (C).
Soluble proteins extracts obtained by sonication of E. coli BL21(DE3) cells carrying pMCR or pMCR-N-C were used for western blot analysis and enzyme activity assay. Representative western blot result from triplicated independent experiments is shown. Enzyme activity assay was performed in Tris-HCl buffer pH7.2 at 37°C. Date represent mean ± standard deviation (N=3).