Part:BBa_K4596001

Nicotinamide cytosine dinucleotide synthetase in E. coli -> Ncds

This enzyme can synthesize the non-natural coenzyme nicotinamide cytosine dinucleotide (NCD) using nicotinamide mononucleotide (NMN) and cytidine triphosphate (CTP) (Wang et al. 2021). This enzyme can be used to synthesize the non-natural coenzyme NCD, which can then be used for more bioorthogonal reactions.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 600
  
• 1000
  COMPATIBLE WITH RFC[1000]

Profile

Name: Ncds-2
Base Pairs: 642 bp
Origin: Escherichia coli, synthetic
Properties: Catalyze the synthesis of non-natural coenzyme nicotinamide cytosine dinucleotide

Characterization

We cloned the Ncds-2 gene into the pet-28(a) vector with a 6xHis tag for easy purification of the Ncds enzyme.

We expressed and purified the recombinant Ncds enzyme in Escherichia coli BL21 and eluted it from a nickel affinity column with a buffer containing 40mM imidazole. The recombinant Ncds enzyme did not elute from the column in a buffer with 30mM imidazole.

Figure 1.Ncds-2 protein gel diagram. Imidazole elution concentrations were 0, 10, 20, 30, 40, and 70 mM for each well on the right side of Marker.

References

Wang, X, Feng, Y, Guo, X, Wang, Q, Ning, S, Li, Q, Wang, J, Wang, L & Zhao, ZK 2021, ‘Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide’, Nature Communications, vol. 12, no. 1, p. 2116.