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University of Tokyo discovers new biosynthetic pathway for terpenoids: Expected to make wide-ranging contributions to drug discovery research in the production of functions beyond those in natural compounds

2022.07.11

Terpenoid compounds are a group of natural organics with the greatest structural diversity in nature and are used in a variety of fields, including pharmaceuticals, fragrances, and resins. In particular, they are very important as a source for searching for drug compound candidates as they contain numerous biologically active compounds. Among them, triterpenes with a carbon number of 30 (C30) are produced in all living organisms across species in microorganisms, plants and animals, and include precursors of biological steroids such as cholesterol, which is important as a component of cell membranes, various steroid hormones and bile acids. To date, the only known biosynthetic pathway for triterpenes is via a 30-carbon (C30) squalene, a further dimerization of 15-carbon (C15) farnesyl diphosphate (FPP), which is formed by the condensation of a 5-carbon (C5) isoprene unit, dimethylallyl diphosphate (DMAPP), with isopentenyl diphosphate (IPP), by prenyl transferase.

A joint research group led by Professor Ikuro Abe, Assistant Professor Takahiro Mori and project researcher Hui Tao of the Graduate School of Pharmaceutical Sciences, the University of Tokyo, Professor Toshiya Senda and project Associate Professor Naruhiko Adachi of the High Energy Accelerator Research Organization (KEK), Wuhan University and the University of Bonn, has conducted a functional analysis of a mold-derived terpene synthase, and were the first in the world to discover a revolutionary novel biosynthetic enzyme that builds the C30 triterpene structure in one step using non-squalene C5 isoprene unit dimethylallyl pyrophosphate (DMAPP) and isopentenyl diphosphate (IPP) as substrates. Their findings were published in the online edition of Nature.

The group's finding is revolutionary in regard to non-squalene enzymes. Specifically, for the purpose of comprehensive analysis of candidate genes for a novel chimeric terpene synthase consisting of two domains, prenyltransferase and terpene cyclase found in a series of fungi (filamentous fungi), the research group caused heterologous expression in yeast hosts and found that novel triterpene compounds were produced in yeast expressing the two different mold-derived terpene synthases TvTS and MpMS, respectively. They then found, as a result of conducting an enzymatic reaction in vitro using the purified recombinant enzyme and analyzing it in detail, these chimeric terpene synthases were revolutionary novel enzymes that construct the C30 triterpene structure simultaneously by the two domains of prenyltransferase and terpene cyclase using C5 unit DMAPP and IPP as substrates.

Furthermore, they identified the reaction mechanisms of the two triterpene synthases in detail by performing enzymatic reactions with substrates marked with stable isotopes. X-ray crystallographic analysis of the terpene cyclase domain of TvTS and cryo-EM structural analysis of the overall structure of MpMS revealed that TvTS and MpMS have active sites large enough to accept the C30 hexaprenyl diphosphate intermediate.

Next, the group created a model of the complex with the substrate, and based on the structure of the active site, examined how hexaprenyl diphosphate binds to the active site by introducing mutations in the amino acid residues of the active site, and proposed a detailed cyclase reaction mechanism in combination with the results of in vitro enzymatic reactions. Furthermore, by searching for similar enzymes and analyzing their functions using a three-dimensional structural model, they discovered a new triterpene synthase, CgCS, and proved that this novel triterpene biosynthesis machinery is widely retained in molds.

This achievement is the discovery of a new biosynthetic pathway and a revolutionary novel enzyme that turns existing thinking on its head. It will have a great academic impact, such as the development of new molecular recognition chemistry and the establishment of a new catalyst concept, and in the future, it is expected to make a broad contribution to drug discovery research, including the creation of new functional molecules that surpass natural products through the redesign of biosynthetic machinery using synthetic biology methods.

Journal Information
Publication: Nature
Title: Discovery of non-squalene triterpenes
DOI: 10.1038/s41586-022-04773-3

This article has been translated by JST with permission from The Science News Ltd.(https://sci-news.co.jp/). Unauthorized reproduction of the article and photographs is prohibited.

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