Characterization of D-xylonate dehydratase YjhG from Escherichia coli
Characterization of D-xylonate dehydratase YjhG from Escherichia coli
复制标题
大肠杆菌 D-木糖酸脱水酶 YjhG 的表征
DOI：
10.1080/21655979.2015.1040208
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发表时间：
2015-07-01
期刊：
影响因子：
4.9
通讯作者：
Xian, Mo
中科院分区：
文献类型：
--
作者：
Jiang, Yudong;Liu, Wei;Xian, Mo
来自大肠杆菌的D-xylonate脱水酶YjhG可以将D-xylonate转化为2-酮-3-脱氧- D-xylonate (KDX)，是1,2,4-丁三醇等化学物质生物合成的关键酶。然而，YjhG的生化特性仍然未知。本研究在氨基脲法的基础上建立了YjhG活性测定方法，其中KDX与氨基脲试剂反应，并进一步采用高分辨率质谱法进行定量。用纯化的his标记的YjhG蛋白体外测定反应条件对YjhG活性的影响。该酶在30℃、pH 8.0条件下活性最高。Mg2+和Mn2+等二价金属离子对YjhG具有活化作用，而Ni2+和Zn2+对YjhG具有抑制作用。在最佳条件下，以d -木酸盐为底物，Km和Vmax分别为4.88 mM和78.62 μM l−1h−1。氨基酸序列比对和催化性能分析表明，YjhG可能是IlvD/EDD家族的成员。本研究结果可为进一步研究YjhG奠定基础，并有利于其在相关化学品的生物合成中的应用。
D-xylonate dehydratase YjhG from Escherichia coli can convert D-xylonate into 2-keto-3-deoxy- D-xylonate (KDX), and is a key enzyme in the biosynthesis of 1,2,4-butanetriol and other chemicals. However, the biochemical properties of YjhG still remain unknown. In this study, the activity assay method for YjhG was established based on semicarbazide method, in which KDX reacts with semicarbazide reagent, and is further quantified by high-resolution mass spectrometry. The effect of reaction conditions on YjhG activity was determined in vitro using purified His-tagged YjhG protein. This enzyme showed maximal activity at 30°C and pH 8.0. Bivalent metal ions such as Mg2+ and Mn2+ activated, whereas Ni2+ and Zn2+ inhibited the activity of YjhG. Under optimal conditions, the Km and Vmax values were 4.88 mM and 78.62 μM l−1h−1, respectively, when using D-xylonate as a substrate. Amino acids sequence alignments and catalytic properties analysis revealed that YjhG might be a member of IlvD/EDD family. Results obtained in this study may lay a foundation for further investigation on YjhG and will benefit its application in biosynthesis of related chemicals.