Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans
Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans
复制标题
单氨基酸突变改变了从副球菌分离的鲨肌醇脱氢酶中 L-葡萄糖和肌醇的底物特异性
DOI：
10.1080/09168451.2019.1702870
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Yajima Shunsuke
中科院分区：
文献类型：
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作者：
Suzuki Mayu;Koubara Kairi;Takenoya Mihoko;Fukano Kazuhiro;Ito Shinsaku;Sasaki Yasuyuki;Nakamura Akira;Yajima Shunsuke
从laevigucosivorans副球菌（Pl-sIDH）中分离出的三叉肌醇脱氢酶显示出广泛的底物特异性：它氧化三叉肌醇和肌醇以及葡萄糖，将葡萄糖转化为葡萄糖-1,5-内酯。根据先前报道的晶体结构，Arg178残基位于催化位点的入口端口，似乎对接受底物很重要。在这里，我们报告了Arg178的作用，通过使用丙氨酸取代突变体进行动力学分析以及确定晶体结构。野生型Pl-sIDH表现出纤维肌醇的活性，其次是肌醇和葡萄糖。相反，R178A突变体消除了这两种肌醇的活性，但保持了与野生型相同程度的葡萄糖活性。根据突变体的晶体结构，Asp191的侧链从底物结合位点翻转。因此，Arg178对于正确定位Asp191发挥其催化活性非常重要。缩写：IDH：肌醇脱氢酶；磅:Luria-Bertani;Kcat：催化剂速率常数；Km：米切里斯常数；NAD：烟酰胺二核苷酸；NADH：烟酰胺二核苷酸还原形式；PDB;蛋白质数据库；PDB条目：6KTJ， 6KTK, 6KTL
scyllo-inositol dehydrogenase, isolated fromParacoccus laeviglucosivorans(Pl-sIDH), exhibits a broad substrate specificity: it oxidizesscyllo- andmyo-inositols as well asl-glucose, convertingl-glucose tol-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity forscyllo-inositol most preferably followed bymyo-inositol andl-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active forl-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities.Abbreviations:IDH: inositol dehydrogenase; LB: Luria-Bertani;kcat: catalyst rate constant;Km: Michaelis constant; NAD: nicotinamide dinucleotide; NADH: nicotinamide dinucleotide reduced form; PDB; Protein Data Bank; PDB entry: 6KTJ, 6KTK, 6KTL