Terpene cyclases are a mechanistically intriguing family of enzymes that catalyze complex, multi-step reactions capable of generating hundreds of structurally 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 beginning to yield a set of physical-chemical rules that govern the relationship between enzyme sequence diversity, catalytic mechanism and sesquiterpene chemical diversity. Structure and sequence comparisons of TEAS in HPS guided the stepwise mutagenesis of several TEAS residues into the corresponding HPS residues, resulting in the identification of 9 mutations that inter-convert TEAS into HPS. Construction of the reciprocal 9 mutant enzyme in HPS effectively inter-converted HPS to TEAS. Together, these results identify a mutational pathway connecting two divergent biosynthetic properties. Interestingly, only 2 of the 9 mutations are in the active site, while the other 7 are in the outer tiers of the protein, some as far away as 15 angstroms from the reaction center. This notable achievement raises several mechanistic and evolutionary questions. Because these mutations were made sequentially, the question remains, are all nine mutations required for inter-converting product selectivity? What combinations, if any, of these nine mutations are sufficient for this property change? Moreover, in a broader evolutionary sense, what is the mechanistic and energetic landscape that links such specificity switches that are encoded by multiple changes at sites throughout a given protein? Towards answering these questions, Structure-based Combinatorial Protein Engineering (SCOPE) was applied to create a mutant library comprising all permutations of nine mutations which inter-convert TEAS into HPS (29 = 512 combinations) representing all possible mutational pathways (9! = 362,880 paths). Purification and characterization of the product profiles each of the 512 library proteins by GC-MS is nearly complete and beginning to answer important fundamental questions raised by the inter-conversion results. Several minimal combinations of the original 9 mutations have been identified which convert TEAS into an “HPS-like” cyclase. Interestingly, several mutants display an alternative biosynthetic property altogether, producing a novel terpene 4-epi-eremophilene as their dominant product. Furthermore, a large class of highly-active mutants displays promiscuous activities. Annotating this enzyme lineage created by SCOPE with the chemical output detected by GC-MS provides the first high-resolution map of the evolutionary pathways underlying the emergence of chemical complexity in terpene metabolism.

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