Grechkin Alexander N, Brühlmann Fredi, Mukhtarova Lucia S, Gogolev Yuri V, Hamberg Mats
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan, 420111 Russia.
Biochim Biophys Acta. 2006 Dec;1761(12):1419-28. doi: 10.1016/j.bbalip.2006.09.002. Epub 2006 Sep 14.
The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.
研究了重组甜瓜（黄瓜）水合过氧化物裂解酶（HPL，CYP74C亚家族）对亚油酸9-氢过氧化物（9-HPOD）的转化。在0℃下短时间（5秒 - 1分钟）孵育，然后快速提取和三甲基硅烷化，使得能够捕获一种新的不稳定产物（半衰期<30秒），即半缩醛（1'E,3'Z）-9-羟基-9-（1',3'-壬二烯氧基）-壬酸。通过气相色谱 - 质谱分析进行鉴定，并通过催化氢化后形成三甲基硅烷基9-三甲基硅氧基-9-壬氧基-壬酸酯以及氘标记实验得到证实。[(18)O(2)-氢过氧基]9-HPOD的两个(18)O原子都并入了半缩醛中。除了半缩醛外，还捕获了三种链裂解产物，即烯醇（1E,3Z）-壬二烯醇以及3(Z)-壬烯醛和9-氧代壬酸的水合物，它们以三甲基硅烷基衍生物的形式存在。[(18)O(2)]9-HPOD中(18)O的掺入动力学提供了有力证据，表明裂解产物起源于半缩醛。亚麻酸和亚油酸13-氢过氧化物用作甜瓜、苜蓿（紫花苜蓿）和番石榴（番石榴）重组HPL的底物，并且在每种情况下，通过捕获技术都可检测到半缩醛和烯醇。所获得的数据表明，CYP74C和CYP74B HPL作为异构酶，将脂肪酸氢过氧化物进行均裂重排形成短寿命的半缩醛，半缩醛分解后产生3(Z)-壬烯醛、3(Z)-己烯醛和其他短链醛。
