Part:BBa_K3711012
curA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
CurA is derived from curcumin-transforming microorganisms isolated from human faeces with a molecular weight of about 82 kDa and consists of two identical subunits. During the purification of the expressed curA enzyme, the activity of curA enzyme was lost during dialysis. However, we found that the addition of NADPH increased the activity, but the addition of NADH did not increase the activity. These findings indicate that the enzyme catalyzes the NADPH-dependent transformation of curcumin. In the curA catalytic reaction, two steps of curcumin metabolism pathway (curcumin→dihydrocurcumin→tetrahydrocurcumin) were found. Under the catalysis of CurA, dihydrocurcumin is first the product, then the substrate. These products are produced from curcumin by reducing the diarylheptatrienone chain which could devastate the color of the hair. The curA does not produce more reduction products than tetrahydrocurcumin, which indicates that curA only catalyzes the reduction of compounds with C=C. In the reaction process, the optimum reaction temperature of curA is 35 ℃. The enzyme showed maximum activity at pH 5.9. Although a large number of different compounds have been tested as potential substrates of curA, curA seems to have a narrow substrate spectrum and preferentially acts on curcumin. Considering substrate specificity and NADPH dependence, curA was named NADPH-dependent curcumin/dihydrocurcumin reductase.
Molecular cloning
The bands of AOX1-α factor-curA-AOX1 Terminator (almost 3000bp), AOX1-α factor-pepACS-AOX1 Terminator (almost 2000bp) and AOX1-α factor-DsbC-AOX1 Terminator (almost 3000bp) from colony PCR are identical to the theoretical lengths of 2875bp, 1987bp and 2722bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these target plasmid had successfully transformed into E.coli.
Using E.coli for amplification, we extract and digest them with Bgl I or Sal I to get linear plasmid, which could be integrated into yeast genome to avoid getting lost while being frozen. Then, concentration of linear plasmid is also applied to achieve higher copy number and higher expression level. Several rounds of electroporation later, we successfully get all the plasmid with AOX1 as promoter into yeast.
The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast.
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