Part:BBa_K1398002

NemA (N-ethylmaleimide reductase)

N-ethylmaleimide reductase, created for use as a Trinitroltoluene and Nitroglycerine degrading protein. NemA is a flavoprotein that primarily catalyses the reduction of N-ethylmaleimide (NEM), which is toxic to cell growth. However, it is also involved in the degradation of other toxic compounds for their reuse in nitrogen metabolism. Some of these compounds include PETN, quinones and chromate. It increases the resistance of organisms to the toxic effects of these compounds. This sequence only encodes for the protein, an attached His (x6) Tag to allow for purification and a double-STOP codon. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression. The protein has been codon-optimised for expression in E. coli.

The Exeter 2014 team used Raman spectroscopy to show the breakdown of Nitroglycerin by NemA:

The Exeter 2014 team also showed that NemA cannot perform this catalytic function without cofactors: FMN, NADH and NADPH.

Analysis of these data allowed the kinetic parameters of Km and Vmax of NemA for nitroglycerin to be calculated as 6 M and 21 umol per mg per minute, respectively.

Full article at: http://2014.igem.org/Team:Exeter/EnzymeValidation

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 NgoMIV site found at 520
  Illegal AgeI site found at 357
  
• 1000
  COMPATIBLE WITH RFC[1000]