Part:BBa_K5136006

CYP199A4 T253D-His tag

Biology

CYP199A4 is a NADH-dependent cytochrome P450 monooxygenase from Rhodopseudomonas palustris cytochrome P450, a heme-dependent enzyme that is a versatile bio-oxidation catalyst for C–X (e.g., X = H, N, S) bond oxidations (1). CYP199A4 can also function as peroxygenase. The engineered CYP199A4 peroxygenases showed good functional group tolerance and preferential O-demethylation at the meta- or para-position, indicating potential O-demethylation of H- and G-type lignin monomers (1).

Usage and design

CYP199A4 can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2). It has been pointed out that position T253 of CYP199A4 can affect the coordination environment of the active center (1). Therefore, we carried out saturation mutagenesis on this site, hoping to screen out enzymes with higher activity.
Partial amino acid sequence of CYP199A4 WT: …AGLDTDVNGI…

Construction

We use pET-28a(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Figure 1 Gene circuit of CYP199A4 T253D-His tag.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1412 bp.

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136006_pET-28a(+).

The pET-28a (+) vectors containing CYP199A4 variants were transformed into E. coli BL21(DE3), and the cells were cultivated in LB medium containing 50 μg/mL kanamycin. The cultures were grown at 37 °C with vigorous shaking (~200 rpm). When the OD₆₀₀ of the cultures reached 0.8~1.0, the temperature was cooled to 20 °C, and the expression was induced by the addition of IPTG (0.2 mM) and δ-aminolevulinic acid hydrochloride (0.5 mM). Following 16-20 h of expression, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 45.8 kDa).

Figure 3 SDS-PAGE analysis of CYP199A4 T253D-His tag protein.

Reference

1. P. Zhao, Y. Jiang, Q. Wang, J. Chen, F. Yao, Z. Cong, Crucial gating residues govern the enhancement of peroxygenase activity in an engineered cytochrome P450 O-demethylase. Chemical Science 15, 8062-8070 (2024).
2. Shen Kui-zhong, Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 150
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 831
  Illegal XhoI site found at 1231
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 150
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 150
  Illegal AgeI site found at 691
  
• 1000
  COMPATIBLE WITH RFC[1000]