Structural Basis of Cyclized C17 Precursor Recognition and Product Formation in Chlororaphen and Grimophan Biosynthesis

Abstract Terpene cyclases (TCs) typically transform acyclic oligoprenyl pyrophosphates through carbocationic cyclization cascades. However, recently discovered bacterial pathways generate compact, cyclized and stereochemically defined noncanonical intermediates through methyltransferases prior to TC catalysis, raising the question of how these unusual substrates are recognized and converted. Here, we report high-resolution crystal structures of two bacterial C17 class I TCs, Pc-ChloS and Vb-GriS, including a complex with a substrate surrogate that defines recognition of the cyclized precursor α-prechlororaphen pyrophosphate (α-PCPP). Combined with mutagenesis and isotope-labeling experiments, these structures provide a molecular framework for chlororaphen and grimophan biosynthesis. Despite highly similar active-site architectures, reciprocal mutagenesis identifies a single Thr/Leu exchange at position 89 as a major determinant of product partitioning. Together with comparisons to other class I TCs, these findings reveal how a conserved scaffold accommodates cyclized terpene precursors, while subtle active-site changes redirect complex carbocation cascades and product formation.