Structural Insights into a Bacterial Terpene Cyclase Fused with Haloacid Dehalogenase-like Phosphatase

Abstract

Terpene cyclases (TCs), consisting of various combinations of α, β, and γ domains, have been extensively studied. Recently, non-canonical enzymes comprising a TCβ domain and a haloacid dehalogenase (HAD)-like domain (referred to as HAD-TCβ) have been discovered. However, their overall structure remains unclear. In this study, we determined the co-crystal structures of drimenol synthase from Aquimarina spongiae (AsDMS), which catalyzes the conversion of farnesyl pyrophosphate (1) into drimenol (2). Crystallographic analyses of the enzyme bound to substrates 1 and drimenyl monophosphate (3) demonstrated that the TCβ domain catalyzes a class II cyclization reaction initiated by protonation, whereas the HAD domain catalyzes a phosphatase-like dephosphorylation reaction dependent on a divalent metal. Crystallographic and gel filtration analyses revealed that AsDMS adopts a dimeric assembly. This dimerization positioned the TCβ and HAD domains to facilitate efficient substrate transfer via electrostatic substrate channeling. Furthermore, to investigate the structure-function relationship of the AsDMS TCβ domain, we used AlphaFold2 to model the structure of the fungal albicanol (4) synthase. Comparative analysis of active-site residues between AsDMS and fungal 4-synthase enabled rational protein engineering, converting AsDMS activity from 2-synthase to 4-synthase. This study provides insights into the biosynthesis of valuable drimane-type sesquiterpenes via targeted mutagenesis.

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Article information

Article type
Edge Article
Submitted
26 Jun 2025
Accepted
27 Jul 2025
First published
28 Jul 2025
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2025, Accepted Manuscript

Structural Insights into a Bacterial Terpene Cyclase Fused with Haloacid Dehalogenase-like Phosphatase

K. Fujiyama, H. Takagi, N. N. Q. Vo, N. Morita, T. Nogawa and S. Takahashi, Chem. Sci., 2025, Accepted Manuscript , DOI: 10.1039/D5SC04719F

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