Coupling the Isopentenol Utilization Pathway and Prenyltransferase for the Biosynthesis of Licoflavanone in Recombinant Escherichia coli.

Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.

Prenylation of aromatic amino acids and plant phenolics by an aromatic prenyltransferase from Rasamsonia emersonii.

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for L-tryptophan and L-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37-55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On L-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. KEY POINTS: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO.

Methods for the preparation and analysis of a bifunctional class II diterpene synthase, copalyl diphosphate synthase from Penicillium fellutanum.

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Maramycin, a Cytotoxic Isoquinolinequinone Terpenoid Produced through Heterologous Expression of a Bifunctional Indole Prenyltransferase/Tryptophan Indole-Lyase in S. albidoflavus.

Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase/tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.

Genome Mining Reveals a UbiA-Type Prenyltransferase Access to Farnesylation of Diketopiperazines.

UbiA-type prenyltransferases (PTases) are significant enzymes that lead to structurally diverse meroterpenoids. Herein, we report the identification and characterization of an undescribed UbiA-type PTase, FtaB, that is responsible for the farnesylation of indole-containing diketopiperazines (DKPs) through genome mining. Heterologous expression of the fta gene cluster and non-native pathways result in the production of a series of new C2-farnesylated DKPs. This study broadens the reaction scope of UbiA-type PTases and expands the chemical diversity of meroterpenoids.

Water-deficit stress induces prenylated stilbenoid production and affects biomass in peanut hairy roots: Exploring the role of stilbenoid prenyltransferase downregulation.

The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes-mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress.

Enzymatic properties and immobilization of a thermostable prenyltransferase from Aspergillus fumigatiaffinis for the production of prenylated naringenin.

Prenyltransferases catalyze the synthesis of prenylated flavonoids, providing these with greater lipid solubility, biological activity, and availability. In this study, a thermostable prenyltransferase (AfPT) from Aspergillus fumigatiaffinis was cloned and expressed in Escherichia coli. By optimizing induction conditions, the expression level of AfPT reached 39.3 mU/mL, which was approximately 200 % of that before optimization. Additionally, we determined the enzymatic properties of AfPT. Subsequently, AfPT was immobilized on carboxymethyl cellulose magnetic nanoparticles (CMN) at a maximum load of 0.6 mg/mg. Optimal activity of CMN-AfPT was achieved at pH 8.0 and 55 °C. Thermostability assays showed that the residual activity of CMN-AfPT was greater than 50 % after incubation at 55 °C for 4 h. Km and Vmax of CMN-AfPT for naringenin were 0.082 mM and 5.57 nmol/min/mg, respectively. The Kcat/Km ratio of CMN-AfPT was higher than that of AfPT. Residual prenyltransferase activity of CMN-AfPT remained higher than 70 % even after 30 days of storage. Further, CMN-AfPT retained 68 % of its original activity after 10 cycles of reuse. Compared with free AfPT, CMN-AfPT showed higher catalytic efficiency, thermostability, metal ion tolerance, substrate affinity, storage stability, and reusability. Our study presents a thermostable prenyltransferase and its immobilized form for the production of prenylated flavonoids in vitro.

Vitamin K converting enzyme UBIAD1 plays an important role in osteogenesis and chondrogenesis in mice.

Dietary vitamin K1 (phylloquinone: PK) and menaquinone (MK-n) are converted to menadione (MD) in the small intestine and then translocated to various tissues where they are converted to vitamin K2 (menaquinone-4: MK-4) by UbiA prenyltransferase domain containing protein 1 (UBIAD1). MK-4 is effective in bone formation and is used to treat osteoporosis in Japan. UBIAD1 is expressed in bone and osteoblasts and shows conversion to MK-4, but the role of UBIAD1 in osteogenesis is unknown. In this study, we investigated the function of UBIAD1 in osteogenesis using a tamoxifen-dependent UBIAD1-deficient mouse model. When UBIAD1 deficiency was induced from the first week of life, the femur was significantly shortened, and bone mineral density (BMD) was reduced. In addition, the expression of bone and chondrocyte matrix proteins and chondrocyte differentiation factors was significantly decreased. In primary cultured chondrocytes, chondrocyte differentiation was significantly reduced by UBIAD1 deficiency. These results suggest that UBIAD1 is an important factor for the regulation of chondrocyte proliferation and differentiation during osteogenesis.

Parallel Evolution of Asco- and Basidiomycete O-Prenyltransferases.

Prenyltransferases (PTs) are involved in the biosynthesis of a multitude of pharmaceutically and agriculturally important plant, bacterial, and fungal compounds. Although numerous prenylated compounds have been isolated from Basidiomycota (mushroom-forming fungi), knowledge of the PTs catalyzing the transfer reactions in this group of fungi is scarce. Here, we report the biochemical characterization of an O- and C-prenylating dimethylallyltryptophan synthase (DMATS)-like enzyme LpTyrPT from the scurfy deceiver Laccaria proxima. This PT transfers dimethylallyl moieties to l-tyrosine at the para-O position and to l-tryptophan at atom C-7 and represents the first basidiomycete l-tyrosine PT described so far. Phylogenetic analysis of PTs in fungi revealed that basidiomycete l-tyrosine PTs have evolved independently from their ascomycete counterparts and might represent the evolutionary origin of PTs acting on phenolic compounds in secondary metabolism.

Structure of the prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum reveals an open hexamer conformation.

Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.

Foldseek reveals a CBGA prenylating enzyme GlyMa_02G168000 from Glycine max.

The present research provides an application for an aromatic prenyltransferase from Glycine max for use in heterologous microorganism expression to generate cannabinoids. The known cannabinoid prenyltransferase CsPT04 was queried in FoldSeek. An enzyme derived from Glycine max known as GLYMA_02G168000, which is a predicted homogentisate solanyltransferase, was identified and found to have affinity for the prenylation of geranyldiphosphate (GPP) and olivetolic acid (OA) to produce cannabigerolic acid (CBGA) and cannabigerol (CBG). The in vitro production of CBGA was accomplished through the heterologous expression of this prenyltransferase in Saccharomyces cerevisiae. After growing the yeast cells, a purified microsomal fraction was harvested, which was rich in the membrane-bound prenyltransferase GlyMa_02G168000. Addition of purified microsomal fraction to a reaction matrix facilitated the successful prenylation of externally supplied OA with GPP, culminating in the production of CBGA. Structural comparisons revealed a notably closer similarity between GLYMA_02G168000 and CsPT04, compared to the similarity of other cannabinoid prenyltransferases with CsPT04. Herein, a novel application for a homogentisate solanyltransferase has been established towards the production of cannabinoids.

Structure Prediction and Genome Mining-Aided Discovery of the Bacterial C-Terminal Tryptophan Prenyltransferase PalQ.

Post-translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing genome mining methods combined with AlphaFold2-based predictions of protein interactions, PalQ , a prenyltransferase responsible for the tryptophan prenylation of RiPPs produced by Paenibacillus alvei, is identified. PalQ differs from cyanobactin prenyltransferases because of its evolutionary relationship to isoprene synthases, which enables PalQ to transfer extended prenyl chains to the indole C3 position. This prenylation introduces structural diversity to the tryptophan side chain and also leads to conformational dynamics in the peptide backbone, attributed to the cis/trans isomerization that arises from the formation of a pyrrolidine ring. Additionally, PalQ exhibited pronounced positional selectivity for the C-terminal tryptophan. Such enzymatic characteristics offer a toolkit for peptide therapeutic lipidation.

Expanding the phenotypic and genotypic spectrum of GGPS1 related congenital muscular dystrophy.

Congenital muscular dystrophies are a group of progressive disorders with wide range of symptoms associated with diverse cellular mechanisms. Recently, biallelic variants in GGPS1 were linked to a distinct autosomal recessive form of muscular dystrophy associated with hearing loss and ovarian insufficiency. In this report, we present a case of a young patient with a homozygous variant in GGPS1. The patient presented with only proximal muscle weakness, and elevated liver transaminases with spared hearing function. The hepatic involvement in this patient caused by a novel deleterious variant in the gene extends the phenotypic and genotypic spectrum of GGPS1 related muscular dystrophy.

Prenyltransferase gene expression reveals an essential role of prenylation for the inflammatory response in human gingival fibroblasts.

BACKGROUND: Prenyltrasferases (PTases) are a class of enzymes known to be responsible for promoting posttranslational modification at the carboxyl terminus of proteins containing a so-called CaaX-motif. The process is responsible for proper membrane localization and the appropriate function of several intracellular signaling proteins. Current research demonstrating the pathomechanistic importance of prenylation in inflammatory illnesses emphasizes the requirement to ascertain the differential expression of PT genes under inflammatory settings, particularly in periodontal disease. METHODS: Telomerase-immortalized human gingival fibroblasts (HGF-hTert) were cultured and treated with either inhibitors of prenylation (PTI) lonafarnib, tipifarnib, zoledronic acid, or atorvastatin at concentrations of 10 µM in combination with or without 10 µg Porphyromonas gingivalis lipopolysaccharide (LPS) for 24 h. Prenyltransferase genes FNTB, FNTA, PGGT1B, RABGGTA, RABGGTB, and PTAR1 as well as inflammatory marker genes MMP1 and IL1B were detected using quantitative real-time polymerase chain reaction (RT-qPCR). Immunoblot and protein immunoassay were used to confirm the results on the protein level. RESULTS: RT-qPCR experiments revealed significant upregulation of IL1B, MMP1, FNTA, and PGGT1B upon LPS treatment. PTase inhibitors caused significant downregulation of the inflammatory cytokine expression. Interestingly, FNTB expression was significantly upregulated in response to any PTase inhibitor in combination with LPS, but not upon LPS treatment only, indicating a vital role of protein farnesyltransferase in the proinflammatory signaling cascade. CONCLUSIONS: In this study, distinct PTase gene expression patterns in pro-inflammatory signaling were discovered. Moreover, PTase inhibiting drugs ameliorated inflammatory mediator expression by a significant margin, indicating that prenylation is a major pre-requisite for innate immunity in periodontal cells.

Regulation of protein prenylation.

Prenyltransferases (PTases) are known to play a role in embryonic development, normal tissue homeostasis and cancer by posttranslationally modifying proteins involved in these processes. They are being discussed as potential drug targets in an increasing number of diseases, ranging from Alzheimer's disease to malaria. Protein prenylation and the development of specific PTase inhibitors (PTIs) have been subject to intense research in recent decades. Recently, the FDA approved lonafarnib, a specific farnesyltransferase inhibitor that acts directly on protein prenylation; and bempedoic acid, an ATP citrate lyase inhibitor that might alter intracellular isoprenoid composition, the relative concentrations of which can exert a decisive influence on protein prenylation. Both drugs represent the first approved agent in their respective substance class. Furthermore, an overwhelming number of processes and proteins that regulate protein prenylation have been identified over the years, many of which have been proposed as molecular targets for pharmacotherapy in their own right. However, certain aspects of protein prenylation, such as the regulation of PTase gene expression or the modulation of PTase activity by phosphorylation, have attracted less attention, despite their reported influence on tumor cell proliferation. Here, we want to summarize the advances regarding our understanding of the regulation of protein prenylation and the potential implications for drug development. Additionally, we want to suggest new lines of investigation that encompass the search for regulatory elements for PTases, especially at the genetic and epigenetic levels.

Hormonal control of promoter activities of Cannabis sativa prenyltransferase 1 and 4 and salicylic acid mediated regulation of cannabinoid biosynthesis.

Cannabis sativa aromatic prenyltransferase 4 (CsPT4) and 1 (CsPT1) have been shown to catalyze cannabigerolic acid (CBGA) biosynthesis, a step that rate-limits the cannabinoid biosynthetic pathway; both genes are highly expressed in flowers. CsPT4 and CsPT1 promoter driven ß-glucuronidase (GUS) activities were detected in leaves of cannabis seedlings, and strong CsPT4 promoter activities were associated with glandular trichomes. Hormonal regulation of cannabinoid biosynthetic genes is poorly understood. An in silico analysis of the promoters identified putative hormone responsive elements. Our work examines hormone-responsive elements in the promoters of CsPT4 and CsPT1 in the context of physiological responses of the pathway to the hormone in planta. Dual luciferase assays confirmed the regulation of promoter activities by the hormones. Further studies with salicylic acid (SA) demonstrated that SA pretreatment increased the expression of genes located downstream of the cannabinoid biosynthetic pathway. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis. The work provides information relevant to plant biology, as we present evidence demonstrating correlations between molecular mechanisms that regulate gene expression and influence plant chemotypes.

Biosynthesis of polyprenylated xanthones in Hypericum perforatum roots involves 4-prenyltransferase.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of 5 variants of xanthone 4-prenyltransferase (HpPT4px) comprising 4 long variants (HpPT4px-v1 to HpPT4px-v4) and 1 short variant (HpPT4px-sh). The full-length sequences of all 5 variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone; 1,3,5-trihydroxyxanthone; and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.

Geranylation of Chalcones by a Fungal Aromatic Prenyltransferase.

Geranylated chalcones mainly exist in plants, and many of them have attracted attention because of their diverse pharmacological and biological activities. Herein, we report geranylation of eight chalcones by the Aspergillus terreus aromatic prenyltransferase AtaPT. Ten new mono-geranylated enzyme products (1G-5G, 6G1, 6G2, 7G, 8G1, and 8G2) were obtained. Most of the products are C-geranylated products with prenyl moieties at ring B. In comparison, plant aromatic prenyltransferases usually catalyze the geranylation at ring A. Therefore, AtaPT can be used complementarily for chalcone geranylation to increase the structural diversity of small molecules. In addition, seven compounds (1G, 3G, 4G, 6G1, 7G, 8G1, and 8G2) exhibited a potential inhibitory effect on α-glucosidase with the IC50 values ranging from 45.59 ± 3.48 to 82.85 ± 2.15 µg/mL. Among them, compound 7G (45.59 ± 3.48 µg/mL) was the most potential α-glucosidase inhibitor, which is about seven times stronger than the positive control acarbose (IC50 = 346.63 ± 15.65 µg/mL).

The functional evolution of architecturally different plant geranyl diphosphate synthases from geranylgeranyl diphosphate synthase.

Terpenoids constitute the largest class of plant primary and secondary metabolites with a broad range of biological and ecological functions. They are synthesized from isopentenyl diphosphate and dimethylallyl diphosphate, which in plastids are condensed by geranylgeranyl diphosphate synthases (GGPPSs) to produce GGPP (C20) for diterpene biosynthesis and by geranyl diphosphate synthases (GPPSs) to form GPP (C10) for monoterpene production. Depending on the plant species, unlike homomeric GGPPSs, GPPSs exist as homo- and heteromers, the latter of which contain catalytically inactive GGPPS-homologous small subunits (SSUs) that can interact with GGPPSs. By combining phylogenetic analysis with functional characterization of GGPPS homologs from a wide range of photosynthetic organisms, we investigated how different GPPS architectures have evolved within the GGPPS protein family. Our results reveal that GGPPS gene family expansion and functional divergence began early in nonvascular plants, and that independent parallel evolutionary processes gave rise to homomeric and heteromeric GPPSs. By site-directed mutagenesis and molecular dynamics simulations, we also discovered that Leu-Val/Val-Ala pairs of amino acid residues were pivotal in the functional divergence of homomeric GPPSs and GGPPSs. Overall, our study elucidated an evolutionary path for the formation of GPPSs with different architectures from GGPPSs and uncovered the molecular mechanisms involved in this differentiation.