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1.
ACS Chem Biol ; 14(9): 2035-2043, 2019 09 20.
Article in English | MEDLINE | ID: mdl-31433159

ABSTRACT

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.


Subject(s)
Acyclic Monoterpenes/chemistry , Diphosphates/chemistry , Diterpenes/chemistry , Enzyme Inhibitors/chemistry , Intramolecular Lyases/antagonists & inhibitors , Polyisoprenyl Phosphates/chemistry , Catalytic Domain , Citrus sinensis/enzymology , Crystallography, X-Ray , Diphosphates/chemical synthesis , Diterpenes/chemical synthesis , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Intramolecular Lyases/chemistry
2.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1033-1034: 9-16, 2016 Oct 15.
Article in English | MEDLINE | ID: mdl-27517522

ABSTRACT

Trehalose is an important disaccharide that is used as a cellular protectant by many different organisms, helping these organisms better survive extreme conditions, such as dehydration, oxidative stress, and freezing temperatures. Methods to detect and accurately measure trehalose from different organisms will help us gain a better understanding of the mechanisms behind trehalose's ability to act as a cellular protectant. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using selected reaction monitoring mode for the detection and quantification of trehalose using maltose as an internal standard has been developed. This assay uses a commercially available LC column for trehalose separation and a standard triple quadrupole mass spectrometer, thus allowing many scientists to take advantage of this simple assay. The calibration curve from 3 to 100µM trehalose was fit best by a single polynomial. This LC-MS/MS assay directly detects and accurately quantifies trehalose, with an instrument limit of detection (LOD) that is 2-1000 times more sensitive than the most commonly-used assays for trehalose detection and quantification. Furthermore, this assay was used to detect and quantify endogenous trehalose produced by Escherichia coli (E. coli) cells, which were found to have an intracellular concentration of 8.5±0.9mM trehalose. This method thus shows promise for the reliable detection and quantification of trehalose from different biological sources.


Subject(s)
Chromatography, Liquid/methods , Protective Agents/analysis , Tandem Mass Spectrometry/methods , Trehalose/analysis , Escherichia coli/chemistry , Escherichia coli/metabolism , Limit of Detection , Linear Models , Reproducibility of Results
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