Dataset Information

Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis.

ABSTRACT: Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2-7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.

SUBMITTER: Xing B

PROVIDER: S-EPMC9814573 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis.

Xing Baiying B Yu Jiahui J Chi Changbiao C Ma Xueyang X Xu Qingxia Q Li Annan A Ge Yuanjie Y Wang Zhengdong Z Liu Tan T Jia Hongli H Yin Fuling F Guo Juan J Huang Luqi L Yang Donghui D Ma Ming M

Communications chemistry 20210930 1

Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2-7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. ...[more]

PMID: 36697656

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Project description:As a major staple food, maize (Zea mays) is critical to food security. Shifting environmental pressures increasingly hamper crop defense capacities, causing expanded harvest loss. Specialized labdane-type diterpenoids are key components of maize chemical defense and ecological adaptation. Labdane diterpenoid biosynthesis most commonly requires the pairwise activity of class II and class I diterpene synthases (diTPSs) that convert the central precursor geranylgeranyl diphosphate into distinct diterpenoid scaffolds. Two maize class II diTPSs, ANTHER EAR 1 and 2 (ZmAN1/2), have been previously identified as catalytically redundant ent-copalyl diphosphate (CPP) synthases. ZmAN1 is essential for gibberellin phytohormone biosynthesis, whereas ZmAN2 is stress-inducible and governs the formation of defensive kauralexin and dolabralexin diterpenoids. Here, we report the biochemical characterization of the two remaining class II diTPSs present in the maize genome, COPALYL DIPHOSPHATE SYNTHASE 3 (ZmCPS3) and COPALYL DIPHOSPHATE SYNTHASE 4 (ZmCPS4). Functional analysis via microbial co-expression assays identified ZmCPS3 as a (+)-CPP synthase, with functionally conserved orthologs occurring in wheat (Triticum aestivum) and numerous dicot species. ZmCPS4 formed the unusual prenyl diphosphate, 8,13-CPP (labda-8,13-dien-15-yl diphosphate), as verified by mass spectrometry and nuclear magnetic resonance. As a minor product, ZmCPS4 also produced labda-13-en-8-ol diphosphate (LPP). Root gene expression profiles did not indicate an inducible role of ZmCPS3 in maize stress responses. By contrast, ZmCPS4 showed a pattern of inducible gene expression in roots exposed to oxidative stress, supporting a possible role in abiotic stress responses. Identification of the catalytic activities of ZmCPS3 and ZmCPS4 clarifies the first committed reactions controlling the diversity of defensive diterpenoids in maize, and suggests the existence of additional yet undiscovered diterpenoid pathways.

Project description:BACKGROUND: Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. RESULTS: Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. CONCLUSIONS: SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

Dataset Information

Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis.

Publications

Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis.

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