Engineering an efficient and enantioselective enzyme for the Morita–Baylis–Hillman reaction

Abstract

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. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative scale transformations, accepts a broad range of aldehyde and enone coupling partners, and is able to promote selective mono-functionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via 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). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in Nature.

Efficient base-catalysed Kemp elimination in an engineered ancestral enzyme

“…Furthermore, most engineered de novo enzymes display very low activities and many rounds of laboratory directed evolution, a highly time-consuming procedure, are often required to bring their catalysis to levels similar to those of modern natural enzymes [37,45-47].…”

“…However, only about 30% of enzymes are metalloenzymes [43,44]and mechanisms for the emergence of new enzymes that do not rely on metal recruitment remain poorly understood. Furthermore, most engineered de novo enzymes display very low activities and many rounds of laboratory directed evolution, a highly time-consuming procedure, are often required to bring their catalysis to levels similar to those of modern natural enzymes [37,[45][46][47].…”

Redesigning Enzymes for Biocatalysis: Exploiting Structural Understanding for Improved Selectivity

“…In a further recent development from the Arnold lab, they report the P450 catalyzed enantioselective ring expansion of aziridines to azetidines (Miller et al, 2022). Other methods to enable new to nature reactions (Leveson-Gower et al, 2019) include utilizing expanded genetic code via amber codon technology and tuning active site electronics (Ortmayer et al, 2020) as well as using Rosetta to design enzymes to carry out new to nature chemistry (Crawshaw et al, 2021).…”

“…The logical progression of these strategies is in silico enzyme design facilitated by, for example, Rosetta. This tool has already been applied by several groups (Crawshaw et al, 2021) (Basanta et al, 2020) (Richter et al, 2011). Both approaches will also generate greater insight into the factors affecting substrate binding and catalytic mechanism which in turn will lead to more predictability.…”

“…Computational methodology will enable a greater understanding of the role of flexible regions and protein dynamics in catalysis. Thus new computational tools and their democratization will, alongside the ongoing protein engineering revolution, lead to the second great revolution in enzyme engineering and is already proving exciting (Büchler et al, 2022) (Crawshaw et al, 2021). This combination will provide a much-needed pool of sustainable catalysts and hopefully a new era in chemical synthesis.…”