Engineering an Efficient and Enantioselective Enzyme for the Morita-Baylis-Hillman Reaction

Rebecca Crawshaw, Amy Crossley, Linus Johannissen, Ashleigh Burke, Sam Hay, Colin Levy, David Baker, Sarah Lovelock, Anthony Green

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The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. To date, this approach has delivered enzymes for a handful of model reactions, selected based on previous achievements with catalytic antibodies. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate valuable chemical transformations for which no natural enzymes or catalytic antibodies are known. Evolutionary optimization of a primitive design afforded a highly efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction, a valuable multi-step carbon-carbon bond forming process. BH32.14 is suitable for preparative scale transformations, accepts a broad range of aldehyde and enone coupling partners, including challenging MBH substrates such as unsaturated lactones, and is able to promote highly selective mono-functionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that the interplay of design and evolution has led to a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen bonding catalysts (e.g. thioureas), which promote a wide range of reactions in organic synthesis. This study makes us optimistic about the prospects of designing enzymes for complex multi-step transformations not observed in Nature, but suggests that innovations in multi-state design protocols are needed to account for conformational changes required for efficient catalysis.
Original languageEnglish
JournalNature Chemistry
Publication statusPublished - 16 Dec 2021

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology


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