Itoh Takafumi, Mikami Bunzo, Hashimoto Wataru, Murata Kousaku
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
J Mol Biol. 2008 Apr 11;377(5):1443-59. doi: 10.1016/j.jmb.2008.01.090. Epub 2008 Feb 12.
The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-D-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an alpha6/alpha6-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (D-mannose) at 1.6 A resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, beta-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, alpha6/alpha6-barrel.
肠炎沙门氏菌假定蛋白YihS的三维结构在共同支架α6/α6桶方面与N-酰基-D-葡糖胺2-表异构酶（AGE）显著相似，尽管YihS的功能仍有待阐明。为了确定YihS的功能，将大肠杆菌和肠炎沙门氏菌的YihS蛋白在大肠杆菌中过表达、纯化并进行表征。发现这两种蛋白均无AGE活性，但具有不依赖辅因子的醛糖-酮糖异构酶活性，参与单糖、甘露糖、果糖和葡萄糖或来苏糖和木酮糖的相互转化。为了阐明YihS的结构/功能关系，我们以1.6埃的分辨率确定了肠炎沙门氏菌YihS突变体（H248A）与底物（D-甘露糖）复合物的晶体结构。这种酶-底物复合物结构是AGE结构家族中的首次展示，它使我们能够识别活性位点残基并推测YihS的反应机制。底物β-D-甘露糖很好地契合活性位点并被该酶特异性识别。YihS与甘露糖C1和O5原子的底物结合位点在结构上与变旋酶的相似，这表明YihS通过His383采用吡喃糖开环过程并使C2位置酸化，在C1位置形成醛。在异构化步骤中，His248作为碱催化剂，负责通过顺式烯二醇中间体将质子从C2转移到C1位置。另一方面，在AGE中，His248被认为是从底物的C2位置提取并重新结合质子。这些发现不仅为YihS反应机制提供了分子层面的见解，也为具有共同支架α6/α6桶的新型碳水化合物活性酶的分子设计提供了有用信息。
