Coding

Part:BBa_K4580000

Designed by: Michael Constant   Group: iGEM23_Cornell   (2023-10-09)

NdmA- 6x his

This gene encodes for the first N-demethylase in the caffeine demethylation pathway within the NdmABCD operon [1]. Attached to it is a 6x-histidine tag, this part is utilized for easy enzyme isolation via affinity chromatography and/ or specific mutation without the other genes within the original operon.

Allows for Escherichia coli cells to degrade caffeine into theobromine and paraxanthine into 7-Methxylxanthine (7-MX). [2]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 915


Usage and Biology

While caffeine is a simple molecule and relatively cheap to purchase, its derivatives hold a medical application that is not easily seen. Studies performed by Dr. Ryan Summers and their team have utilized a multi-strand method of biosynthetically creating 7-methxylanthine, one of caffeine's derivatives [2]. The usage for this compound comes from its medical application within ophthalmology. Clinical trials have revealed that 7-methxylanthine (or 7-MX for short) has a correlation on moderating severe forms of myopia (nearsightedness) on patients where surgeries, such as LASIK, cannot be performed. By producing this compound biologically, the traditional and expensive method of chemical synthesis can be evaded by utilizing the type III caffeine demethylation pathway shown below [3].

Figure 1: Type III Caffeine Demethylation Pathway

NdmA specifically is the first N-demethylase within the pathway that starts the pathway. The important organic products of focus that NdmA generates are theobromine ( 3,7-dimethylxanthine) converted from caffeine (1,3,7-trimethylxanthine) and 7-MX from paraxanthine (1,7-dimethylxanthine). To complete this, a co-enzyme found from NdmD passes a H+ proton from NAD(P)H to help drive the reaction.

After designing BBa_K4580000, a gel confirmation was done along with BBa_K4580004 to check for noticeable size difference which was successful.

Figure 2: Gel Confirmation of BBa_K4580000 and BBa_K4580004

Functionality

Catalytic Efficiency

Substrate Product Km (uM) kcat (min^-1) kcat/Km (min^-1 / uM)
1. Caffeine Theobromine 37 190 5.1
2. Paraxanthine 7-methylxanthine 53 130 2.5

This data comes from Dr. Ryan Summers in [2].

References

[1] UT Austin. (2012). BBA_K734000. NdmABCD operon. https://parts.igem.org/Part:BBa_K734000

[2] Summers, R. M., Louie, T. M., Yu, C. L., Gakhar, L., Louie, K. C., & Subramanian, M. (2012). Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. Journal of bacteriology, 194(8), 2041–2049. https://doi.org/10.1128/JB.06637-11

[3] Caffeine Demethylation Pathway Type III. MetaCyc caffeine degradation III (bacteria, via Demethylation). (n.d.). http://vm-trypanocyc.toulouse.inra.fr/META/NEW-IMAGE?type=PATHWAY&object=PWY-6538&detail-level=4

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