d'Andréa S, Canonge M, Beopoulos A, Jolivet P, Hartmann M A, Miquel M, Lepiniec L, Chardot T
Institut National de la Recherche Agronomique, UMR Chimie Biologique, CBAI INRA INA PG, BP1, F-78850 Thiverval Grignon, France.
Biochimie. 2007 Feb;89(2):222-9. doi: 10.1016/j.biochi.2006.09.013. Epub 2006 Oct 11.
In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.
在之前的一项研究中，我们提供了证据，证明拟南芥中由At5g50600编码的一种蛋白质存在于油体（OBs）中[P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot，拟南芥生态型WS油体的蛋白质组成，植物生理学与生物化学42 (2004) 501 - 509]。使用特异性抗体和蛋白质组学技术，我们目前证实了这种蛋白质的存在，它是短链类固醇脱氢酶还原酶超家族的成员。我们已经测定了它对各种类固醇（胆固醇、脱氢表雄酮、皮质醇、皮质酮、雌二醇、雌酮）以及NAD(P)(H)的活性，无论是在纯化的油体中还是作为纯化的细菌表达嵌合体。两种酶系统（从拟南芥种子中纯化的油体以及嵌合酶）对雌二醇（17β - 羟基类固醇）都表现出羟基类固醇脱氢酶（HSD）活性，使用NAD⁺或NADP⁺，NADP⁺是首选的辅因子。用皮质醇或皮质酮（11β - 羟基类固醇）观察到低水平的活性，但无论使用何种辅因子，胆固醇和脱氢表雄酮（3β - 羟基类固醇）都不是底物。两种酶来源都发现了相似的活性谱。纯化的油体还被发现能够用NADPH催化雌酮还原（17β - 酮类固醇还原酶活性）。拟南芥油体中存在的这种酶可归类为NADP⁺依赖性11β - 、17β - 羟基类固醇脱氢酶/17β - 酮类固醇还原酶。这种酶可能对应于由At5g50600编码的AtHSD1。然而，其生理作用和底物仍有待确定。
