# Insights into taxadiene synthase catalysis and promiscuity facilitated by mutability landscape and molecular dynamics

## TL;DR

This study explores how taxadiene synthase works and how it can make different products, using modeling and simulations to understand its function and potential for engineering.

## Contribution

The study introduces a mutability landscape and molecular dynamics to uncover the catalytic and promiscuous behavior of taxadiene synthase.

## Key findings

- Mutability landscape analysis identified residues linked to TXS promiscuity, such as V584, Q609, V610, and Y688.
- Certain mutants increased the production of a more favorable Taxol precursor, taxa-4(20),11(12)-diene.
- Molecular dynamics simulations revealed residues involved in carbocation stabilization and cyclization during TXS catalysis.

## Abstract

Protein modeling, carbocation docking, and molecular dynamics along with structure-based mutability landscapes provided insight into taxadiene synthase catalysis (first step of the anticancer Taxol biosynthesis), protein structure–function correlations, and promiscuity.

Plant terpenes belong to one of the largest and most diverse classes of natural products. This diversity is driven by the terpene synthase enzyme family which comprises numerous different synthases, several of which are promiscuous. Taxadiene synthase (TXS) is a class I diterpene synthase that catalyzes the first step in the biosynthesis pathway of the diterpene Taxol, an anticancer natural product produced by the Taxus plant. Exploring the molecular basis of TXS catalysis and its promiscuous potential garnered interest as a necessary means for understanding enzyme evolution and engineering possibilities to improve Taxol biosynthesis. A catalytically active closed conformation TXS model was designed using the artificial intelligence system, AlphaFold, accompanied by docking and molecular dynamics simulations. In addition, a mutability landscape of TXS including 14 residues was created to probe for structure–function relations. The mutability landscape revealed no mutants with improved catalytic activity compared to wild-type TXS. However, mutations of residues V584, Q609, V610, and Y688 showed high degree of promiscuity producing cembranoid-type and/or verticillene-type major products instead of taxanes. Mechanistic insights into V610F, V584M, Q609A, and Y688C mutants compared to the wild type revealed the trigger(s) for product profile change. Several mutants spanning residues V584, Q609, Y688, Y762, Q770, and F834 increased production of taxa-4(20),11(12)-diene which is a more favorable substrate for Taxol production compared to taxa-4(5),11(12)-diene. Finally, molecular dynamics simulations of the TXS reaction cascade revealed residues involved in ionization, carbocation stabilization, and cyclization ushering deeper understanding of the enzyme catalysis mechanism.

The online version contains supplementary material available at 10.1007/s00425-024-04363-9.