Coevolution-based protein engineering of alcohol dehydrogenase at distal sites enables enzymatic compatibility with substrate diversity and stereoselectivity.

Chiral alcohols with various substituents and functional groups are attractive synthesizers in many fields. Biocatalysts have attracted great interest for their use in " sustainable chemistry". However, substrate specificity of enzymes limits their widespread use as "generalists" in biocatalysis. In addition, engineering enzymes for simultaneously improving catalytic efficiency and stereoselectivity for structurally diverse substrates is a contemporary challenge. Inspired by naturally occurring coevolution of residues dedicated to a particular function and clustered together in space, we applied coevolution-based engineering to the alcohol dehydrogenase CpRCR from Candida parapsilosis to identify distal sites which can synergistically improve the catalytic properties of diverse substrates. Five variants were developed by clustering the coupling strength and structure of coevolutionary sites which showed improved (up to 28-fold) catalytic efficiency with high stereoselectivity toward 16 structurally diverse substrates (aryl ketones, heterocyclic ketones and β-ketoesters). In particular, for substrate 2-acetylpyridine, the specific activity of K191L/D216H is 12-fold higher than the previously reported highest activity of alcohol dehydrogenase. Theses distal mutations do not directly modify the active center but rather modulate catalytic capacity in various allosteric ways favoring substrate diversity. This study provides a broadly applicable strategy for protein engineering and expanded the applications of biocatalyst on value-added chemicals.