scholarly journals CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway

2019 ◽  
Vol 60 (11) ◽  
pp. 2510-2522 ◽  
Author(s):  
Keylla U Bicalho ◽  
Mariana M Santoni ◽  
Philipp Arendt ◽  
Cleslei F Zanelli ◽  
Maysa Furlan ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Quinone Methide ◽  
Second Step ◽  
Nuclear Magnetic Resonance Analysis ◽  
Root Bark ◽  
Biosynthesis Pathway ◽  
Oxidosqualene Cyclase ◽  
Resonance Analysis ◽  
Maytenus Ilicifolia

Abstract The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

New Tirucallane-Type Triterpenoids from Guarea guidonia

Planta Medica ◽  
2018 ◽  
Vol 84 (09/10) ◽  
pp. 716-720 ◽  
Author(s):  
Vanessa Hernandez ◽  
Marinella De Leo ◽  
Roberta Cotugno ◽  
Alessandra Braca ◽  
Nunziatina De Tommasi ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Resolution Mass Spectrometry ◽  
Lactone Ring ◽  
Nuclear Magnetic Resonance Analysis ◽  
Spectroscopic Methods ◽  
One Dimensional ◽  
Resonance Analysis ◽  
Aerial Parts ◽  
Guarea Guidonia ◽  
Cytotoxic Responses

AbstractThe aerial parts of Guarea guidonia afforded three new tirucallane-type triterpenoids: 3,4-seco-tirucalla-4(28),8(9),24(25)-trien-7α,11α-dihydroxy-21,23-epoxy-3,11-olide, named guareolide (1), 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21-hydroxy-21,23-epoxy-3-oic acid, named guareoic acid A (2), and 3,4-seco-tirucalla-4(28),7(8),24(25)-trien-21,23-epoxy-3-oic acid, named guareoic acid B (3), of which 1 possessed an unusual seven-membered lactone ring. Seven known terpenes were also isolated and characterized as flindissone, 7-acetyldihydronomilin, picroquassin E, boscartol C, and cneorubins A, B, and X. Their structures were determined by spectroscopic methods including one-dimensional and two-dimensional nuclear magnetic resonance analysis and high-resolution mass spectrometry. The isolates were investigated for their potential cytotoxic activity on Jurkat, HeLa, and MCF7 cancer cell lines. Flindissone and compound 2 showed an antiproliferative activity in all cell lines. Further studies revealed that flindissone, the most active compound, induced in Jurkat and HeLa cells both cytostatic and cytotoxic responses.

scholarly journals Biosynthetic Pathways of Inositol and Glycerol Phosphodiesters Used by the Hyperthermophile Archaeoglobus fulgidus in Stress Adaptation

2006 ◽  
Vol 188 (23) ◽  
pp. 8128-8135 ◽  
Author(s):  
Nuno Borges ◽  
Luís G. Gonçalves ◽  
Marta V. Rodrigues ◽  
Filipa Siopa ◽  
Rita Ventura ◽  
...  
Keyword(s):  
Inositol Phosphate ◽  
Compatible Solutes ◽  
Regulatory Mechanisms ◽  
Archaeoglobus Fulgidus ◽  
Nuclear Magnetic Resonance Analysis ◽  
Biosynthetic Pathways ◽  
Inositol 1 ◽  
Resonance Analysis ◽  
Intermediate Metabolites

ABSTRACT Archaeoglobus fulgidus accumulates di-myo-inositol phosphate (DIP) and diglycerol phosphate (DGP) in response to heat and osmotic stresses, respectively, and the level of glycero-phospho-myo-inositol (GPI) increases primarily when the two stresses are combined. In this work, the pathways for the biosynthesis of these three compatible solutes were established based on the detection of the relevant enzymatic activities and characterization of the intermediate metabolites by nuclear magnetic resonance analysis. The synthesis of DIP proceeds from glucose-6-phosphate via four steps: (i) glucose-6-phosphate was converted into l-myo-inositol 1-phosphate by l-myo-inositol 1-phosphate synthase; (ii) l-myo-inositol 1-phosphate was activated to CDP-inositol at the expense of CTP; this is the first demonstration of CDP-inositol synthesis in a biological system; (iii) CDP-inositol was coupled with l-myo-inositol 1-phosphate to yield a phosphorylated intermediate, 1,1′-di-myo-inosityl phosphate 3-phosphate (DIPP); (iv) finally, DIPP was dephosphorylated into DIP by the action of a phosphatase. The synthesis of the two other polyol-phosphodiesters, DGP and GPI, proceeds via the condensation of CDP-glycerol with the respective phosphorylated polyol, glycerol 3-phosphate for DGP and l-myo-inositol 1-phosphate for GPI, yielding the respective phosphorylated intermediates, 1X,1′X-diglyceryl phosphate 3-phosphate (DGPP) and 1-(1X-glyceryl) myo-inosityl phosphate 3-phosphate (GPIP), which are subsequently dephosphorylated to form the final products. The results disclosed here represent an important step toward the elucidation of the regulatory mechanisms underlying the differential accumulation of these compounds in response to heat and osmotic stresses.

scholarly journals Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens

2001 ◽  
Vol 67 (10) ◽  
pp. 4701-4707 ◽  
Author(s):  
Søren F. Lassen ◽  
Jens Breinholt ◽  
Peter R. Østergaard ◽  
Roland Brugger ◽  
Andrea Bischoff ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Nuclear Magnetic Resonance Analysis ◽  
Initial Attack ◽  
Ph Optimum ◽  
Resonance Analysis ◽  
Hydrolysis Of ◽  
Temperature Optima ◽  
Expression Libraries ◽  
Basidiomycete Fungi

ABSTRACT Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed inAspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U · mg, of protein−1 and were capable of hydrolyzing phytate down tomyo-inositol monophosphate. Surprisingly, 1H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26 ).

Synthesis and characterization of lignin–melamine–formaldehyde resin

2016 ◽  
Vol 30 (9) ◽  
pp. 1255-1266
Author(s):  
Amadou Diop ◽  
Kokou Adjallé ◽  
Benjamin Boëns ◽  
Daniel Montplaisir ◽  
Simon Barnabé
Keyword(s):  
Exothermic Peak ◽  
Formaldehyde Resin ◽  
Nuclear Magnetic Resonance Analysis ◽  
Hydroxyl Groups ◽  
Gravimetric Analysis ◽  
Dsc Analysis ◽  
Melamine Formaldehyde ◽  
Scanning Calorimetry ◽  
Resonance Analysis

Lignin–melamine–formaldehyde (LMF) resin was prepared by three steps: (i) tosylation of lignin, (ii) synthesis of lignin-melamine (LM) copolymer, and (iii) formation of methylol LM. The synthesized resins were characterized by Fourier transform infrared (FTIR) spectroscopy and phosphorous 31 nuclear magnetic resonance analysis. The curing parameters of LMF resin were determined by differential scanning calorimetry (DSC) and thermal gravimetric analysis. The yield of tosylation is 80%. The FTIR spectrum of tosylated lignin shows the presence of two new bands at 1171 and 1370 cm−1. The formation of the LM was demonstrated by the disappearance of both bands and appearance of the absorbances at 3115, 3312, 3415, and 3470 cm−1 corresponding to the stretching vibrations of primary and secondary amine. The peaks observed at 147.0 and 148.5 ppm are attributed to the new aliphatic hydroxyl groups formed by the methylolation of LM. One exothermic peak was observed in the DSC analysis indicating a one cross-linking reaction.

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