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Identifying and manipulating evolutionary determinants linking catalytic specificities in enzymes of specialized metabolism

Terpene cyclases catalyze multistep reactions generating generating 100s of diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Investigating the structural basis for the divergent biosynthetic properties of plant sesquiterpene cyclases Nictotiana tabacum 5‐epi‐aristolochene synthase (TEAS) and Hyoscyamus muticus premnaspirodiene synthase (HPS) is yielding physical‐chemical rules governing the relationship between sequence diversity, catalytic mechanism and chemical diversity. Comparisons of TEAS and HPS guided the stepwise mutagenesis of TEAS into HPS, resulting in the identification of 9 mutations that inter‐convert TEAS into HPS. Construction of the reciprocal 9 mutant enzyme in HPS inter‐converted HPS to TEAS. Structure‐based Combinatorial Protein Engineering (SCOPE) created a mutant library comprising all permutations of the 9 mutations which inter‐convert TEAS into HPS (512 combinations). Purification and biochemical characterization of each of the 512 library proteins answers fundamental questions raised by the inter‐conversion results. Several minimal combinations of the original nine mutations have been identified which convert TEAS into an “HPS‐like” cyclase. Interestingly, several mutants display an alternative biosynthetic property and produce a novel terpene 4‐epi‐eremophilene as their dominant product. Furthermore, a large class of highly‐active mutants displays promiscuous activities. Annotating the enzyme lineages created by SCOPE with their chemical output provides the first high resolution map of the evolutionary pathways underlying the emergence of chemical complexity in terpene metabolism.