From Corals to Labs: Understanding Bifluorene Synthase for Bioactive Compound Production

Bifloran, also known as cellulatan, is characterized by a distinct 6,6-ball framework and prenyl side chains, and presents a wide range of biological activities, including anti-inflammatory, antimalarial and antitumor effects. These compounds are commonly found in marine corals, sponges and plants.

Despite the identification of several biflorene terpene synthases, the catalytic mechanisms and structural basis of the formation of marine biflorene scaffolds remain largely unknown. Furthermore, there is only one crystal structure of coral terpene synthases obtained so far, limiting our understanding of marine coral terpenoid biosynthesis.

The study published in Science Advances discovered several coral terpene synthases, led by Xu Baofu of the Chinese Academy of Sciences (CAS) Shanghai Materia Media Research Institute (SIMM), Guo Yuuewei of SIMM, Wu Ruibo of Sun Yat-Sen University, and Shanghai Institute of Immunity of Enming of the Ensive of the Immungy of the Inced of the Inctiont, and fully elucidated the catalytic mechanism of the biflorene synthase PCTS1.

Researchers have discovered six terpene synthases (TSSs) containing the biflorene synthase PCTS1, focusing on genomic analysis of Sea Whip, Paramuricea clavate. This discovery has enabled detailed mechanistic studies of marine biflorene scaffold formation.

Further experiments using deuterium and fluorine labeling revealed the cyclization pathway of PCTS1. During this process, GGPP isomerized to form GLPP, introducing a 2Z double bond, followed by 1,10 cyclization and 1,3 hydride shift, resulting in closure of the 1,6 ring.

This path continues with two additional 1,2 hydride shifts, eventually culminating in deprotonation. This catalytic pathway disproves previous hypotheses suggesting that two 1,3-water nodal shifts occurred during the process.

Through crystallization experiments, researchers determined the crystal structure of PCTS1. This not only deepens understanding of the biflorene synthase PCTS1, but also facilitates the calculation of quantum/molecular mechanics (QM/mm).

These calculations confirmed the validity of the cyclopropane 2,6 cyclization and opening that occur between the two 1,2 water permeation shifts prior to final deprotonation. In addition, they have largely revealed the role of pyrophosphate as a base responsible for alopecia. This is a difficult aspect of the initial mechanism proposal that is difficult to confirm through labeling the experiment.

Further mutational studies have identified a 10-membered intermediate shunt product from mutant PCTS1I254A that supports the cyclization mechanism starting with the 1,10-cyclization of PCTS1. Subsequent mutagenesis experiments created a variety of unique terpene scaffolds that match the proposed carbon cation intermediates from isotope labeling and QM/MM studies.

Engineering directed towards this mechanism of PCTS1 expanded the diversity of terpene scaffolds and enhanced the potential biocatalysts available for terpene synthesis.

These findings enhance our understanding of coral biphoran formation mechanisms. This study provides a solution to the longstanding challenge of sourcing bioactive compounds from marine corals by utilizing synthetic biological approaches for efficient heterogeneous production.