Redmond T M, Gentleman S, Duncan T, Yu S, Wiggert B, Gantt E, Cunningham F X
Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2001 Mar 2;276(9):6560-5. doi: 10.1074/jbc.M009030200. Epub 2000 Nov 22.
We have identified from mouse the first mammalian beta-carotene 15,15'-dioxygenase (beta-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived precursors. beta-CD is related to the retinal pigment epithelium-expressed protein RPE65 and belongs to a diverse family that includes the plant 9-cis-epoxycarotenoid dioxygenase and bacterial lignostilbene dioxygenases. beta-CD expression in Escherichia coli cells engineered to produce beta-carotene led to the accumulation of all-trans-retinal at the expense of beta-carotene, confirming that beta-CD catalyzed the central cleavage of this vitamin A precursor. Purified recombinant beta-CD protein cleaves beta-carotene in vitro with a V(max) of 36 pmol of retinal/mg of enzyme/min and a K(m) of 6 microm. Non-provitamin A carotenoids were also cleaved, although with much lower activity. By Northern analysis, a 2.4-kilobase (kb) message was observed in liver, kidney, small intestine, and testis, tissues important in retinoid/carotenoid metabolism. This message encoded a 63-kDa cytosolic protein expressed in these tissues. A shorter transcript of 1.8 kb was found in testis and skin. Developmentally, the 2.4-kb mRNA was abundant at embryonic day 7, with lower expression at embryonic days 11, 13, and 15, suggesting a critical role for this enzyme in gastrulation. Identification of beta-CD in an accessible model organism will create new opportunities to study vitamin A metabolism.
我们从小鼠中鉴定出了首个哺乳动物β-胡萝卜素15,15'-双加氧酶（β-CD），它是发育和代谢过程中的一种关键酶，控制着维生素A从植物来源前体的从头进入。β-CD与视网膜色素上皮表达蛋白RPE65相关，属于一个多样的家族，该家族包括植物9-顺式环氧类胡萝卜素双加氧酶和细菌木质芪双加氧酶。在经过基因工程改造以产生β-胡萝卜素的大肠杆菌细胞中，β-CD的表达导致全反式视黄醛的积累，而β-胡萝卜素减少，这证实了β-CD催化了这种维生素A前体的中心裂解。纯化的重组β-CD蛋白在体外裂解β-胡萝卜素，其V(max)为36 pmol视黄醛/毫克酶/分钟，K(m)为6微摩尔。非维生素A类胡萝卜素也能被裂解，不过活性要低得多。通过Northern分析，在肝脏、肾脏、小肠和睾丸这些在类视黄醇/类胡萝卜素代谢中起重要作用的组织中观察到了一条2.4千碱基（kb）的信息。这条信息编码了一种在这些组织中表达的63 kDa胞质蛋白。在睾丸和皮肤中发现了一条1.8 kb的较短转录本。在发育过程中，2.4 kb的mRNA在胚胎第7天丰富，在胚胎第11、13和15天表达较低，这表明该酶在原肠胚形成中起关键作用。在一种易于研究的模式生物中鉴定出β-CD将为研究维生素A代谢创造新的机会。
