Part:BBa_K3853009

his-tag-SpyTag-AAO

Aryl alcohol oxidase (AAO) is an enzyme containing flavin-adenine-dinucleotide (FAD). This is the version with SpyTag. In order to constructed our multi-enzyme complex, we introduced the SpyTag/SpyCatcher system. We added SpyTag to the N-terminus of each protein through 10 × ELP, which is a oligopeptide linker that does not affect the function of the protein it attaches to, as described in the literature^[1]. So this part can be combined with dCas9-SpyCatcher through covalent isopeptide action, thereby being immobilized on dsDNA. His-tag was added to purify the protein. We use BBa_K3853053 to construct the expression system to express and purify the protein.

Biology

Aryl alcohol oxidase (AAO, EC 1.1.3.7), a member of the glucose-methanol-choline oxidase / dehydrogenase (GMC) superfamily, is an enzyme containing flavin-adenine-dinucleotide (FAD) that catalyze the oxidation of aromatic and aliphatic allylic primary alcohols to the corresponding aldehydes while reducing molecular oxygen to H2O2(The mechanism has shown in Fig. 1).

References

[1] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chemical communications (Cambridge, England) 56, 4950-4953, doi:10.1039/d0cc01174f (2020).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 699
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 412
  Illegal BamHI site found at 1411
  Illegal BamHI site found at 1798
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]