Part:BBa_K4275010
MHETase-t
MHETase-t is a dockerin-fused variant of free MHETase (BBa_K4275009). The enzyme shares all the common sequences in the CDS with the former[1], except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of MHETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.
Figure 1 The 3D structure of the protein predicted by Alphafold2.
Usage and Biology
The artificially-designed MHETase - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K. marxianus via ScGPI, or binds to E.coli's Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase-5-dockerin(BBa_K4275011) and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 123
Illegal NgoMIV site found at 237
Illegal NgoMIV site found at 693
Illegal AgeI site found at 190
Illegal AgeI site found at 307 - 1000COMPATIBLE WITH RFC[1000]
References
1. Knott, Brandon C. et al. "Characterization And Engineering Of A Two-Enzyme System For Plastics Depolymerization". Proceedings Of The National Academy Of Sciences, vol 117, no. 41, 2020, pp. 25476-25485. Proceedings Of The National Academy Of Sciences, https://doi.org/10.1073/pnas.2006753117.
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