Blée E, Schuber F
Institute de Biologie Moléculaire des Plantes (CNRS UPR 406), DECM, Institut de Botanique, Strasbourg, France.
J Biol Chem. 1992 Jun 15;267(17):11881-7.
Soluble epoxide hydrolase purified from soybean catalyzes trans-addition of water across the oxirane ring of cis-9,10-epoxystearic acid with inversion of configuration at the attacked carbon, yielding threo-9,10-dihydroxystearic acid. Kinetic analyses of the progress curves, obtained at low substrate concentrations (i.e. [S] much less than Km), and determination of the enantiomeric excess of the residual substrate by chiral-phase high-performance liquid chromatography at different reaction times, indicate that the epoxide hydrolase hydrates preferentially cis-9R, 10S-epoxystearic acid (V/Km ratio, approximately 20). Interestingly, this enantiomer is obtained by epoxidation of oleic acid catalyzed by peroxygenase, a hydroperoxide-dependent oxidase, we have previously described in soybean (Blée, E., and Schuber, F. (1990) J.Biol. Chem. 265, 12887-12894). For the epoxide hydrolase to show high enantioselectivity there must be a free carboxylic acid functionality on the substrate which probably influences its positioning within the active site. This selectivity, which in principle can be used for kinetic resolution of the cis-9,10-epoxystearic acid enantiomers, is much reduced with methyl cis-9,10-epoxystearate. 18O-Labeling experiments indicate that water attacks both cis-9,10-epoxystearic acid enantiomers on the oxirane carbon which has the S-chirality. Results show that soybean epoxide hydrolase produces exclusively threo-9R,10R-dihydroxystearic acid, i.e. a naturally occurring metabolite in higher plants. cis-9,10-Epoxy-18-hydroxystearic acid, a cutin monomer, was a poorer substrate of the epoxide hydrolase than 9,10-epoxystearic acid (V/Km ratio for the preferred enantiomers, approximately 19). From a physiological point of view, peroxygenase and this newly described epoxide hydrolase could be responsible, in vivo, for the biosynthesis of a class of oxygenated fatty acid compounds known to be involved in cutin monomers production and in plant defense mechanisms.
从大豆中纯化得到的可溶性环氧化物水解酶催化水跨顺式-9,10-环氧硬脂酸的环氧乙烷环进行反式加成，在被攻击的碳原子处构型翻转，生成苏式-9,10-二羟基硬脂酸。在低底物浓度（即[S]远小于Km）下获得的反应进程曲线的动力学分析，以及通过手性相高效液相色谱在不同反应时间测定残留底物的对映体过量，表明环氧化物水解酶优先水合顺式-9R,10S-环氧硬脂酸（V/Km比值约为20）。有趣的是，这种对映体是由我们之前在大豆中描述的过氧合酶（一种氢过氧化物依赖性氧化酶）催化油酸环氧化得到的（布莱，E.，和舒伯，F.（1990年）《生物化学杂志》265，12887 - 12894））。为了使环氧化物水解酶表现出高对映选择性，底物上必须有一个游离羧酸官能团，这可能会影响其在活性位点内的定位。这种选择性原则上可用于顺式-9,10-环氧硬脂酸对映体的动力学拆分，但顺式-9,10-环氧硬脂酸甲酯会使其大大降低。18O标记实验表明，水攻击具有S-手性环氧乙烷碳上的两种顺式-9,10-环氧硬脂酸对映体。结果表明，大豆环氧化物水解酶仅产生苏式-9R,10R-二羟基硬脂酸，即高等植物中天然存在的一种代谢产物。角质单体顺式-9,10-环氧-18-羟基硬脂酸是环氧化物水解酶比9,10-环氧硬脂酸更差的底物（优选对映体的V/Km比值约为19）。从生理学角度来看，过氧合酶和这种新描述的环氧化物水解酶在体内可能负责一类已知参与角质单体生成和植物防御机制的氧化脂肪酸化合物的生物合成。
