Identification and Characterization of Bifunctional Drimenol Synthases of Marine Bacterial Origin

Abstract

Natural drimane-type sesquiterpenes, including drimenol, display diverse biological activities. These active compounds are distributed in plants and fungi; however, their accumulation in bacteria remains unknown. Consequently, bacterial drimane-type sesquiterpene synthases remain to be characterized. Here, we report five drimenol synthases (DMSs) of marine bacterial origin, all belonging to the haloacid dehalogenase (HAD)-like hydrolase superfamily with the conserved DDxxE motif typical of class I terpene synthases and the DxDTT motif found in class II diterpene synthases. They catalyze two continuous reactions: the cyclization of farnesyl pyrophosphate (FPP) into drimenyl pyrophosphate and dephosphorylation of drimenyl pyrophosphate into drimenol. Protein structure modeling of the characterized Aquimarina spongiae DMS (AsDMS) suggests that the FPP substrate is located within the interdomain created by the DDxxE motif of N-domain and DxDTT motif of C-domain. Biochemical analysis revealed two aspartate residues of the DDxxE motif that might contribute to the capture of the pyrophosphate moiety of FPP inside the catalytic site of AsDMS, which is essential for efficient cyclization and subsequent dephosphorylation reactions. The middle aspartate residue of the DxDTT motif is also critical for cyclization. Thus, AsDMS utilizes both motifs in the reactions. Remarkably, the unique protein architecture of AsDMS, which is characterized by the fusion of a HADlike domain (N-domain) and a terpene synthase β domain (C-domain), significantly differentiates this new enzyme. Our findings of the first examples of bacterial DMSs suggest the biosynthesis of drimane sesquiterpenes in bacteria and shed light on the divergence of the structures and functions of terpene synthases.

Structure-guided product determination of the bacterial type II diterpene synthase Tpn2

“…These findings suggest that there is more biosynthetic diversity than what is currently known for bacterial diterpenoids, e.g., ent -CPP may not always be transformed into the polycyclic kauranes, atisanes, and pimaranes of currently known bacterial diterpenoids. Add this to the recent discoveries of a monodomain type II TS from cyanobacteria 55 and a bifunctional sesqui-TS that belongs to the haloacid dehalogenase-like hydrolase superfamily 56 and the terpenoid biosynthetic potential of bacteria is likely severely underestimated.…”

Discovery, Structure, and Mechanism of a Class II Sesquiterpene Cyclase

“…TcNudix1, a Nudix hydrolase from Tanacetum cinerariifolium, removes the terminal phosphate group of chrysanthemyl diphosphate in chrysanthemol biosynthesis; RhNUDX1 converts geranyl diphosphate into geranyl monophosphate to facilitate scent production in rose. , Thus, this clustering implicated A0A2P2GK84 and F8JSS1 as monofunctional class II TCs that provide cyclized prenyl diphosphates for subsequent hydrolysis, as seen in drimenol biosynthesis by AoDMS and AcDMS (Figure b). , In addition, these two enzymes share 57 and 64% sequence identity and similarity with each other, respectively, but show very low sequence identity (<20%) with any of the known DMSs of fungi or plant origin (Table S3). − Therefore, we prioritized these two putative STCs for functional characterization.…”

“…A marine bacterial HAD-like DMS was discovered from Aquimarina spongiae (AsDMS) using AoDMS as a genetic beacon during the preparation of this manuscript. 20 In this study, we identified two DPP synthases from Streptomyces showdoensis and Streptomyces cattleya, SsDMS and ScDMS, respectively. Structural characterization of SsDMS using X-ray crystallography led to the determination that these enzymes possess the well-known βγ-didomain structure of canonical class II TCs, an architecture previously unseen in STCs (Figure 1c).…”

“…These examples encouraged us to tap bacterial genomes to identify class II STCs. A marine bacterial HAD-like DMS was discovered from Aquimarina spongiae (AsDMS) using AoDMS as a genetic beacon during the preparation of this manuscript …”

Structure-Guided Product Determination of Bacterial Type II Diterpene Synthases

“…[55,56] These findings suggest that there is more biosynthetic diversity than what is currently known for bacterial diterpenoids, e.g., ent-CPP may not always be transformed into the polycyclic kauranes, atisanes, and pimaranes of currently known bacterial diterpenoids. Add this to the recent discoveries of a monodomain type II TS from cyanobacteria [57] and a bifunctional sesqui-TS that belongs to the haloacid dehalogenase-like hydrolase superfamily [58] and the terpenoid biosynthetic potential of bacteria is likely severely underestimated.…”