Part:BBa_K4169016

produce DMADH

After expressing, it'll produce dimethylamine dehydrogenase (DMADH). The enzyme DMADH is an iron–sulfur flavoprotein which catalyses the oxidative demethylation of dimethylamine (DMA) to methylamine and formaldehyde:
(CH₃)₂N + H₂O → CH₃NH + CH₂O +2H⁺ + 2e^-[1].

Metabolic Pathway

This enzyme is a complex iron-sulfur flavoprotein that transfers electrons to the soluble flavoprotein known as electron transferring flavoprotein[2]. It couldn't work extracellular isolated.

Figure 1.Pathways for dimethylamine metabolism in bacteria.

Protein Molecular Structures

Dimethylamine dehydrogenase(DMADH) exist as dimers.

Figure 2.Protein molecular structures of dimethylamine dehydrogenase.

Engineering Success

We performed SDS-PAGE to identify that trimethylamine dehydrogenase can be expressed. Because trimethylamine dehydrogenase (TMADHexist as dimers, the protein molecular weight would double. So, protein molecular weight of TMADH is 164.9kDa.

Figure 1. Control is E. coli BL21 without dmd. dmd is induced E. coli BL21 with dmd.

We cultivated E. coli BL21 containing dmd and E. coli BL21 without dmd (Blank) for about 3 hours (OD600 0.6~0.8). Then they were induced by 4mM theophylline for 9 hours. After adjusting the density of two tubes of bacteria and making them almost have no difference, we added some DMA into bacteria cultures to make the concentration of substrate DMA 5×10-5mol/L and continued to cultivate them. Take samples before we add DMA, and add DMA for 0 min, 10 min, 20min, 3h, 6h, 9h.

Results shows that although DMATH can be expressed, we can't confirm it could work in E. coli BL21.

Figure 2. Concentration Changes of Metabolism Substrate DMA

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 897
Illegal AgeI site found at 1291
1000
COMPATIBLE WITH RFC[1000]