Coding
CYP

Part:BBa_K5291042:Design

Designed by: Wendan Zheng   Group: iGEM24_BNUZH-China   (2024-09-26)


CYPY96F


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 312
    Illegal BamHI site found at 392
    Illegal BamHI site found at 1221
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 109
    Illegal NgoMIV site found at 277
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The CYPY96F mutant offers distinct advantages for the oxidation of simple alkanes. This engineered variant of the cytochrome P450cam enzyme has been modified to enhance its catalytic activity, resulting in a significant increase in NADH turnover rates and product formation rates, which are approximately 100-fold higher than those of the wild-type enzyme. When undertaking the detailed design of the CYPY96F sequence, a multitude of design considerations were meticulously addressed to ensure the success of the engineered enzyme. The primary goal was to structurally modify the active site by substituting Tyrosine with Phenylalanine at position 96, thereby increasing its hydrophobicity to better accommodate hydrophobic substrates such as alkanes. This alteration was expected to enhance substrate binding and reduce uncoupling, which is a common issue in catalytic cycles that can lead to inefficiency. The mutation was also intended to improve the enzyme's catalytic efficiency without compromising its stability, solubility, or proper folding, including the essential incorporation of the heme group. Furthermore, considerations regarding the enzyme's expression levels in a host organism, its biocompatibility and safety for potential applications, and the scalability of its production were all integral to the design process.


Source

CYPY96F is a variant of the cytochrome P450cam enzyme, which originally comes from a genomic sequence. The parent enzyme, P450cam, is naturally found in the bacterium Pseudomonas putida.

References

Stevenson, J.-A., Westlake, A.C.G., Whittock, C. & Wong, L.-L. The Catalytic Oxidation of Linear and Branched Alkanes by Cytochrome P450cam. Journal of the American Chemical Society 118, 12846-12847 (1996).