Part:BBa_K5136020

SfmD WT

Biology

SfmD is a heme-dependent enzyme in the biosynthetic pathway of saframycin A, which is a heme-dependent enzyme that is a versatile bio-oxidation catalyst for C–X (e.g., X = H, N, S) bond oxidations.
SfmD in Streptomyces lavendulae was previously reported as a heme peroxidase with an atypical peroxidase activity of hydroxylating 3-Me-l-Tyr or l-Tyr using hydrogen peroxide as oxidant (1).

Usage

SfmD 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).

Construction

We use pET28a(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3). The positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Figure 1 Gene circuit of His tag-SfmD WT.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1340 bp.

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136020_pET-28a(+).

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C, the 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 (39.0 kDa).

Figure 3 SDS-PAGE analysis of His tag-SfmD WT protein.

Reference

1.Shin I, Davis I, Nieves-Merced K, Wang Y, McHardy S, Liu A. A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study. Chem Sci. 12, 3984-3998 (2021).
2.Kui-zhong, S. Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 231
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 894
  Illegal NgoMIV site found at 1068
  
• 1000
  COMPATIBLE WITH RFC[1000]