Multi-mechanistic channeling within a biocatalytic cascade for the production of 1,3-diaminopropane
Described herein is a one-pot, four-enzyme cascade of enzymes, three bound to quantum dots with one enzyme free in solution, for the conversion in vitro of fumarate to 1,3-diaminopropane. The cascade operates via two distinctly different enzymatic channeling mechanisms which simultaneously function to increase the overall rate. The first three enzymes of the pathway (AspB->LysC->Asd) were able to engage in channeling in a nanoparticle displayed format, but addition of the last two enzymes to this pathway in this format (AspB->LysC->Asd->Dat->Ddc) did not result in complete channeling through the entire pathway to the final diaminopropane product. Surprisingly, replacement of the last two enzymes (Dat->Ddc) with a naturally occurring fused Dat-Ddc hybrid (Daba) provided for full channeling in this system (AspB->LysC->Asd->Daba).
1 . A method of conducting a cascade reaction, comprising:
providing an enzymatic cascade comprising (1) metallic quantum dots (QDs) having a plurality of enzymes bound thereto, the plurality of enzymes comprising three enzymes comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and (2) a fourth enzyme comprising the amino acid sequence of SEQ ID NO: 6 not bound to the QDs;
contacting the enzymatic cascade with fumarate; and
allowing a reaction to proceed so that each of the plurality of enzymes acts in succession to produce 1,3-diaminopropane from the fumarate.
2 . The method of claim 1 , wherein the reaction is conducted in the presence of an enzyme comprising the amino acid sequence of SEQ ID NO: 7.
3 . An enzymatic cascade comprising (1) metallic quantum dots (QDs) having a plurality of enzymes bound thereto, the plurality of enzymes comprising three enzymes comprising the amino acid sequences of SEQ ID NOS: 1, 2, and 3, respectively, and (2) a fourth enzyme comprising the amino acid sequence of SEQ ID NO: 6 not bound to the QDs.
4 . The enzymatic cascade of claim 3 further comprising an enzyme comprising the amino acid sequence of SEQ ID NO: 7.