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

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Products Chemistry

Background:

• Pyrenulic acids are cytotoxic polyketides from Vietnamese lichens with complex fused cyclic structures.
• The biosynthesis of these natural products was not fully understood.

Purpose of the Study:

• To identify and characterize the biosynthetic gene cluster for pyrenulic acids.
• To elucidate the enzymatic mechanisms involved in constructing the fused cyclic ether core structure.

Main Methods:

• Genome sequencing and bioinformatics analysis to identify the pyrenulic acid (pya) gene cluster.
• Heterologous expression of genes in *Aspergillus nidulans* for functional analysis of enzymes.
• Recombinant enzyme purification, *in vitro* activity assays, and computational studies (DFT, molecular dynamics, site-directed mutagenesis) for PyaF.

Main Results:

• The pyrenulic acid (pya) biosynthetic gene cluster was identified.
• Cytochrome P450s (PyaJ, PyaG) and an α/β hydrolase (PyaF) were characterized.
• PyaF was shown to catalyze successive epoxide ring-opening and cyclization reactions forming a fused bicyclic diether skeleton.
• Computational and mutagenesis studies identified an unusual catalytic serine tetrad and general bases in PyaF crucial for regioselectivity.

Conclusions:

• The study elucidated the key enzymes and mechanisms in pyrenulic acid biosynthesis.
• Understanding PyaF's catalytic mechanism provides insight into controlling regioselectivity in enzymatic epoxide ring-opening cascades for fused cyclic ether formation.