Vinigrol Tricyclic Scaffold Biosynthesis Employs an Atypical Terpene Cyclase and a Multipotent Cyclization Cascade.

Enzymatic Anti-Baldwin Ring-Closure Cascade for Fused Bicyclic Ether Formation.

Pyrenulic acids are cytotoxic polyketides isolated from the ascomycete Pyrenula sp. derived from Vietnamese lichen that are characterized by complex fused cyclic core structures. Genome sequencing, in silico sequence analysis, and RT-PCR studies identified the pyrenulic acid (pya) biosynthetic gene cluster. Based on a functional analysis of the enzymes by expression of each gene in a heterologous host using Aspergillus nidulans, we discovered two cytochrome P450s PyaJ and PyaG that effect epoxidation and hydroxylation of the alkyl chain terminal, respectively, and an α/β hydrolase PyaF that constructs a 6- and 7-membered fused bicyclic diether skeleton by catalyzing successive epoxide ring-opening 6-endo and 7-endo cyclization reactions. To elucidate the detailed mechanism of pyrenulic acid formation, we obtained PyaF as a recombinant enzyme and performed an in vitro experiment, which confirmed catalysis by PyaF of the cyclization reaction. In addition, we performed alignment analysis of PyaF with α/β hydrolases with known functions, as well as an in-depth computational study. In-depth computational analyses of the cyclization reaction pathways with density functional theory quantum mechanics and detailed characterization of PyaF by Chai-1-based protein structure modeling with molecular dynamics simulations and site-specific mutagenesis predicted the active amino acid residues of this serine α/β hydrolase to be an unusual catalytic serine tetrad involving Ser170, Asn342, Asp314, and His169, with Tyr255 and His284 acting as general bases to facilitate opening of the epoxides. Our study provides insight into how regioselectivity of enzymatic anti-Baldwin epoxide ring-opening cascades for the formation of a fused cyclic ether structure is controlled.