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New terpene synthase discovery by RIKEN: High catalytic activity originating from marine bacteria


A research team consisting of Unit Leader Shunji Takahashi and Postdoctoral Researcher Nhu Ngoc Quynh Vo, Natural Product Biosynthesis Research Unit, RIKEN Center for Sustainable Resource Science (CSRS), Dr. Yuta Nomura (postdoctoral researcher at the time of the research; now Assistant Professor at Saitama University), Biomolecular Characterization Unit, Technology Platform Division, CSRS, and their colleagues, has explored the enzyme genes that produce terpenoids using bacteria protein databases, and discovered new sesquiterpene synthases of marine bacterial origin, which effectively produce drimenol (a bicyclic drimane-type sesquiterpene) from farnesyl pyrophosphate (FPP).

Based on the amino acid sequence of a sesquiterpene synthase obtained from the filamentous fungus Aspergillus oryzae (AstC), the research team searched for similar enzyme genes from bacteria protein databases. Moreover, from their amino acid sequence analysis, the team identified six candidates in which important DDxxE and DxDTT motifs, which are common to terpene synthases, are conserved.

To analyze the functions of these enzyme genes, the team prepared expression vectors using artificially synthesized DNA and ensured the enzymes were expressed in E. coli. They carried out reaction analysis using purified enzymes and multiple substrates, and as a result, confirmed that five enzymes were drimenol synthases (DMS) that produce drimenol from farnesyl pyrophosphate (FPP). Of these, DMS derived from the marine bacteria Aquimarina spongiae (AsDMS) demonstrated high catalytic activity when compared to plant-derived DMS. These outcomes show that, of the enzymes discovered on this occasion, AsDMS is the most suited to the realization of rapid drimenol production at low cost.

Moreover, when the team created a structural model of AsDMS, they found that the substrate (FPP) was located in the interdomains of the enzyme's N-terminal and C-terminal protein structures. To verify the required region responsible for catalytic activity, they prepared enzymes that introduced site-directed mutations in amino acids assumed to be present in the active site, and analyzed the reactions. In these enzymes, the two motifs of DDxxE and DxDTT make up the active site, and it was suggested that they are associated with continuous reactions concerning the production of drimenol through the cyclization of FPP to drimenyl pyrophosphate (an intermediate) and the dephosphorylation of drimenyl pyrophosphate.

The team also carried out phylogenetic tree analysis to investigate the evolutionary origin of different AsDMS domains. The results anticipated that AsDMS evolved from haloacid dehalogenase (HAD)-like hydrolase and terpene synthase β, and is a new terpene synthase created from a fusion of two functional domains.

Unit Leader Takahashi commented, "The AsDMS that we have discovered through this study is a new enzyme that is made up of a terpene synthase β domain and HAD-like hydrolase domain, which evolved from different enzyme origins. We hope to discover terpenoid synthases with new functions through further genome scanning research in the future."

■ Terpenoids, Sesquiterpenes: Terpenoid is the general term for a natural organic compound that is made up of five-carbon-atom isoprene units. These are also known as terpenes. They are used as pharmaceuticals, agrochemicals, antibacterial agents, fragrances and food additives. Sesquiterpenes are compounds derived from farnesyl pyrophosphate (FPP), made up of three isoprene units (and 15 carbon atoms). They have diverse structures based on their cyclic form.

■ Motif: An amino acid sequence that is important to enzyme reaction, a conserved domain between enzymes with similar functions.

Journal Information
Publication: ACS Chem. Biol.
Title: Identification and Characterization of Bifunctional Drimenol Synthases of Marine Bacterial Origin
DOI: 10.1021/acschembio.2c00163

This article has been translated by JST with permission from The Science News Ltd.( Unauthorized reproduction of the article and photographs is prohibited.

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