Gough W H, VanOoteghem S, Sint T, Kedishvili N Y
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
J Biol Chem. 1998 Jul 31;273(31):19778-85. doi: 10.1074/jbc.273.31.19778.
We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (</=73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3alpha-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD+ and NADH are the preferred cofactors, with apparent Km values 250-1500 times lower than those for NADP+ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent Ki values of 5.8 and 3.5 microM, respectively. All-trans-retinol bound to cellular retinol-binding protein (type I) exhibits a similar Ki value of 3.6 microM. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD+ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3alpha-androstane-diol.
我们报道了短链脱氢酶/还原酶超家族一个新成员的cDNA序列及其催化特性。从人肝脏cDNA文库中分离得到的1134个碱基对的cDNA编码一种317个氨基酸的蛋白质，即视黄醇脱氢酶4（RoDH-4），它与大鼠的全反式视黄醇脱氢酶RoDH-1、RoDH-2和RoDH-3以及小鼠的顺式视黄醇/雄激素脱氢酶具有最强的相似性（同一性≤73%）。RoDH-4的mRNA在成体肝脏中丰富，在那里它被翻译成RoDH-4蛋白，蛋白质印迹分析证明该蛋白与微粒体膜相关。在胎儿肝脏和肺中检测到大量的RoDH-4信息。使用杆状病毒系统在Sf9昆虫细胞的微粒体中表达的重组RoDH-4，将全反式视黄醇和13-顺式视黄醇氧化为相应的醛，并分别将3α-羟基类固醇雄烷二醇和雄酮氧化为二氢睾酮和雄烷二酮。NAD+和NADH是优选的辅因子，其表观Km值比NADP+和NADPH的表观Km值低250 - 1500倍。全反式视黄醇和13-顺式视黄醇抑制RoDH-4催化的雄酮氧化，其表观Ki值分别为5.8和3.5微摩尔。与细胞视黄醇结合蛋白（I型）结合的全反式视黄醇表现出相似的Ki值3.6微摩尔。未结合配体的细胞视黄醇结合蛋白对RoDH活性没有影响。柠檬醛和无环类异戊二烯也作为RoDH-4活性的抑制剂。乙醇没有抑制作用。因此，我们已经鉴定并表征了一种甾醇/视黄醇氧化短链脱氢酶/还原酶，它优选NAD+并将全反式视黄醇识别为底物。RoDH-4可能有助于两种强大的基因表达调节剂的生物合成：由视黄醇生成视黄酸，以及由3α-雄烷二醇生成二氢睾酮。
