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 <i>Aquimarina spongiae</i> (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 <i>via</i> 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 <i>via</i> targeted mutagenesis.