128 research outputs found
Recommended from our members
Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases
No full textPlants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products
Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate
No full textTrehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce beta-D-glucose 1-phosphate (beta-Glc1P) and D-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40 degrees C, and was stable in the pH range 5.0-8.0 and at up to 30 degrees C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, beta-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, D-mannose 6-phosphate (Man6P). From beta-Glc1P and Man6P, a novel sugar phosphate, alpha-D-Glcp-(1 1)-alpha-D-Manp6P, was synthesized with 51% yield
Efficient one-pot enzymatic synthesis of trehalose 6-phosphate using GH65 α-glucoside phosphorylases
No full textTrehalose 6-phosphate (Tre6P) is an important intermediate for trehalose biosynthesis. Recent researches have revealed that Tre6P is an endogenous signaling molecule that regulates plant development and stress responses. The necessity of Tre6P in physiological studies is expected to be increasing. To achieve the cost-effective production of Tre6P, a novel approach is required. In this study, we utilized trehalose 6-phosphate phosphorylase (TrePP) from Lactococcus lactis to produce Tre6P. In the reverse phosphorolysis by the TrePP, 91.9 mM Tre6P was produced from 100 mM β-glucose 1-phosphate (β-Glc1P) and 100 mM glucose 6-phosphate (Glc6P). The one-pot reaction of TrePP and maltose phosphorylase (MP) enabled production of 65 mM Tre6P from 100 mM maltose, 100 mM Glc6P, and 20 mM inorganic phosphate. Addition of β-phosphoglucomutase to this reaction produced Glc6P from β-Glc1P and thus reduced requirement of Glc6P as a starting material. Within the range of 20-469 mM inorganic phosphate tested, the 54 mM concentration yielded the highest amount of Tre6P (33 mM). Addition of yeast increased the yield because of its glucose consumption. Finally, from 100 mmol maltose and 60 mmol inorganic phosphate, we successfully achieved production of 37.5 mmol Tre6P in a one-pot reaction (100 mL), and 9.4 g Tre6P dipotassium salt was obtained
Function of Plant DExD/H-Box RNA Helicases Associated with Ribosomal RNA Biogenesis
No full textRibosome biogenesis is a highly complex process that requires several cofactors, including DExD/H-box RNA helicases (RHs). RHs are a family of ATPases that rearrange the secondary structures of RNA and thus remodel ribonucleoprotein complexes. DExD/H-box RHs are found in most organisms and play critical roles in a variety of RNA-involved cellular events. In human and yeast cells, many DExD/H box RHs participate in multiple steps of ribosome biogenesis and regulate cellular proliferation and stress responses. In plants, several DExD/H-box RHs have been demonstrated to be associated with plant development and abiotic stress tolerance through their functions in modulating pre-rRNA processing. In this review, we summarize the pleiotropic roles of DExD/H-box RHs in rRNA biogenesis and other biological functions. We also describe the overall function of the DExD/H-box RH family in ribosome biogenesis based on data from human and yeast
How can cold hardening be possible for a cold-deprevid tree? An experimental validation for a physiological model for frost hardiness
No full textabsen
How can cold hardening be possible for a cold-deprevid tree? An experimental validation for a physiological model for frost hardiness
No full textabsen
- …
