Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding.
Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding.
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DOI：
10.1016/j.lfs.2009.06.005
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发表时间：
2009-08
期刊：
影响因子：
6.1
通讯作者：
T. Miura;T. Nishinaka;T. Terada
中科院分区：
文献类型：
--
作者：
T. Miura;T. Nishinaka;T. Terada
目的单体羰基还原酶1（CBR 1）和3（CBR 3）是短链脱氢酶/还原酶超家族的成员，并利用NADPH作为辅酶代谢内源性和外源性化合物。CBR 3与CBR 1相比，对NADPH的Km值更高，羰基还原酶活性更有限，尽管它们在氨基酸序列水平上彼此高度同源。在本研究中，我们调查的起源不同的属性的酶的分析使用几个嵌合enzymes.Main方法sharr-plot分析的氨基酸序列进行，结果，两个低同一性区域之间的人CBR 1和CBR 3被发现：这些被指定为N-末端低同一性区域（LirN）和C-末端低同一性区域（LirC;底物结合区）。我们在遗传学上构建了嵌合酶，同时专注于这些regions.Key发现具有CBR 3的LirN的嵌合CBR 1（CBR 1 LirN 3）表现出CBR 1样活性，但可能由于微结构域的结构改变而导致辅酶亲和力低，而包含CBR 3的LirC的嵌合CBR 1（CBR 1 LirC 3）与CBR 3在酶活性上相似。此外，CBR 3LirC 1在酶活性和辅酶结合方面与CBR 1相似。底物结合环区是人CBR 1和CBR 3在催化和辅酶结合特性方面差异的起源。
AIMSMonomeric carbonyl reductase 1 (CBR1) and 3 (CBR3) are members of the short-chain dehydrogenase/reductase superfamily, and metabolize endogenous and xenobiotic compounds using NADPH as a coenzyme. CBR3 exhibits a higher Kmvalue toward NADPH and more limited carbonyl reductase activities than CBR1, although they are highly homologous to each other in amino acid sequence levels. In the present study, we investigated the origin of the different properties of the enzymes by analyses using several chimeric enzymes.MAIN METHODSHarr-plot analysis of the amino acid sequences was conducted and as a result, two low-identity regions between human CBR1 and CBR3 were found: these were designated as the N-terminal low-identity region (LirN) and the C-terminal low-identity region (LirC; the substrate-binding region). We genetically constructed chimeric enzymes while focusing on these regions.KEY FINDINGSChimeric CBR1 possessing LirN of CBR3 (CBR1LirN3) exhibited CBR1-like activities but a low coenzyme affinity probably due to a structural alteration in a micro domain, whereas chimeric CBR1 including LirC of CBR3 (CBR1LirC3) was enzymatically similar to CBR3. Furthermore, CBR3LirC1 was similar to CBR1 in both enzymatic activities and coenzyme binding.SIGNIFICANCEThese results suggested that LirC, i.e., the substrate-binding loop region, is the origin of the difference between human CBR1 and CBR3 in both catalytic and coenzyme-binding properties.