posted on 2024-02-12, 13:35authored byBaolong Jin, Kangwei Xu, Juan Guo, Ying Ma, Jian Yang, Nianhang Chen, Tao Zeng, Jian Wang, Jianing Liu, Mei Tian, Qing Ma, Haiyan Zhang, Reuben J. Peters, Guanghong Cui, Ruibo Wu, Luqi Huang
Terpenoids are an intriguing class of natural products
with diverse
structures and biological activities whose complexity stems in large
part from terpene synthases (TPSs). These enzymes catalyze carbocationic
cascade reactions wherein the groups responsible for quenching the
final carbocation are generally not well-known. IrKSL3a and IrTPS2
from Isodon rubescens share 98% sequence
homology but use distinct quenching strategies, with IrKSL3a catalyzing
direct deprotonation to generate the olefin isopimaradiene while IrTPS2
adds water to yield the hydroxylated nezukol. In this work, we discovered
a threonine and serine that hydrogen-bond the water to be added in
IrTPS2. Site-directed mutagenesis and multiscale QM/MM simulations
of modeled structures further reveal that the binding of this water
is blocked by the introduction of a β-methyl-containing side
chain in a neighboring residue. From these insights, it was then possible
to engineer IrKSL3a to generate nezukol, with other new hydroxylated
products also observed. Inspired by these mechanistic insights into
the functional plasticity of IrKSL3a and IrTPS2, we explored the plausible
evolutionary relationship of these kaurene synthase-like (KSL) TPSs,
as well as prospective utilization of these plasticity sites discovered
in IrTPS2/IrKSL3a. Such experiments with a variety of more phylogenetically
distant KSLs demonstrated that these residues are necessary but not
sufficient to efficiently introduce such an addition of water, emphasizing
the selective pressure underlying the extended evolutionary process
for the production of nezukol by IrTPS2.