Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy
| Autor: | Lucia S. Mukhtarova, Fredi Brühlmann, Bulat I. Khairutdinov, Alexander N. Grechkin, Mats Hamberg |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Lipid Peroxides Magnetic Resonance Spectroscopy Stereochemistry Linoleic acid Isomerase 01 natural sciences Gas Chromatography-Mass Spectrometry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Oxidoreductase Molecular Biology Aldehyde-Lyases Plant Proteins chemistry.chemical_classification biology Molecular Structure Fatty acid Cytochrome P450 Cell Biology Nuclear magnetic resonance spectroscopy Cucurbitaceae 030104 developmental biology Enzyme chemistry Linoleic Acids biology.protein Hemiacetal 010606 plant biology & botany |
| Zdroj: | Biochimica et biophysica acta. Molecular and cell biology of lipids. 1863(10) |
| ISSN: | 1879-2618 |
| Popis: | Hydroperoxide lyases (HPLs) of the CYP74 family (P450 superfamily) are widely distributed enzymes in higher plants and are responsible for the stress-initiated accumulation of short-chain aldehydes. Fatty acid hydroperoxides serve as substrates for HPLs; however, details of the HPL-promoted conversion are still incompletely understood. In the present work, we report first time the micropreparative isolation and the NMR structural studies of fatty acid hemiacetal (TMS/TMS), the short-lived HPL product. With this aim, linoleic acid 9(S)‑hydroperoxide (9(S)‑HPOD) was incubated with recombinant melon hydroperoxide lyase (CmHPL, CYP74C2) in a biphasic system of water/hexane for 60 s at 0 °C, pH 4.0. The hexane layer was immediately decanted and vortexed with a trimethylsilylating mixture. Analysis by GC–MS revealed a major product, i.e. the bis-TMS derivative of a hemiacetal which was conclusively identified as 9‑hydroxy‑9‑[(1′E,3′Z)‑nonadienyloxy]‑nonanoic acid by NMR-spectroscopy. Further support for the hemiacetal structure was provided by detailed NMR-spectroscopic analysis of the bis-TMS hemiacetal generated from [13C18]9(S)‑HPOD in the presence of CmHPL. The results obtained provide incontrovertible evidence that the true products of the HPL group of enzymes are hemiacetals, and that the short-chain aldehydes are produced by their rapid secondary chain breakdown. Therefore, we suggest replacing the name “hydroperoxide lyase”, which does not reflect the factual isomerase (intramolecular oxidoreductase) activity, with “hemiacetal synthase” (HAS). |
| Databáze: | OpenAIRE |
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