Plant hydroperoxide-cleaving enzymes (CYP74 family) function as hemiacetal synthases: Structural proof of hemiacetals by NMR spectroscopy |
| |
Authors: | Lucia S Mukhtarova Fredi Brühlmann Mats Hamberg Bulat I Khairutdinov Alexander N Grechkin |
| |
Institution: | 1. Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia;2. Firmenich S.A., Corporate R&D, Route des Jeunes 1, CH-1211 Geneva, Switzerland;3. Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden |
| |
Abstract: | 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). |
| |
Keywords: | HPL hydroperoxide lyase HAS hemiacetal synthase AOS allene oxide synthase DES divinyl ether synthase EAS epoxyalcohol synthase 9?HPOD Me methyl TMS trimethylsilyl COSY correlation spectroscopy HSQC heteronuclear single quantum coherence spectroscopy HMBC heteronuclear multiple-bond correlation spectroscopy TOCSY total correlation spectroscopy Hydroperoxide isomerase Hydroperoxide lyase Hemiacetal synthase Cytochrome P450 CYP74 clan |
本文献已被 ScienceDirect 等数据库收录! |
|