Open AccessSubstrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade
Author(s) -
Matthew J. Kummer,
Yoo Seok Lee,
Mengwei Yuan,
Bassam Alkotaini,
Jun Zhao,
Emmy Blumenthal,
Shelley D. Minteer
Publication year - 2021
Publication title -
jacs au
Language(s) - English
Resource type - Journals
ISSN - 2691-3704
DOI - 10.1021/jacsau.1c00180
Subject(s) - linker , chemistry , fusion , combinatorial chemistry , catalysis , protein engineering , enzyme , substrate (aquarium) , alcohol dehydrogenase , cascade , biocatalysis , cationic polymerization , catalytic efficiency , active site , fusion protein , folding (dsp implementation) , biophysics , biochemistry , reaction mechanism , organic chemistry , computer science , recombinant dna , biology , chromatography , ecology , philosophy , linguistics , engineering , gene , electrical engineering , operating system
Substrate channeling, where an intermediate in a multistep reaction is directed toward a reaction center rather than freely diffusing, offers several advantages when employed in catalytic cascades. Here we present a fusion enzyme comprised of an alcohol and aldehyde dehydrogenase, that is computationally designed to facilitate electrostatic substrate channeling using a cationic linker bridging the two structures. Rosetta protein folding software was utilized to determine an optimal linker placement, added to the truncated termini of the proteins, which is as close as possible to the active sites of the enzymes without disrupting critical catalytic residues. With improvements in stability, product selectivity (90%), and catalyst turnover frequency, representing 500-fold increased activity compared to the unbound enzymes and nearly 140-fold for a neutral-linked fusion enzyme, this design strategy holds promise for making other multistep catalytic processes more sustainable and efficient.