Jing Xiaoran, Wang Xinye, Zhang Wenli, An Jianhong, Luo Pengjie, Nie Yao, Xu Yan
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology and State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China.
China National Center for Food Safety Risk Assessment, NHC Key Laboratory of Food Safety Risk Assessment, 37 Guangqu Road, Beijing 100022, China.
ACS Omega. 2019 May 9;4(5):8350-8358. doi: 10.1021/acsomega.9b00983. eCollection 2019 May 31.
Hydroxyl amino acids have tremendous potential applications in food and pharmaceutical industries. However, available dioxygenases are limited for selective and efficient hydroxylation of free amino acids. Here, we identified a 2-oxoglutarate-dependent dioxygenase from by gene mining and characterized the encoded protein (PH1). PH1 was estimated to have a molecular weight of 29 kDa. The optimal pH and temperature for its l-proline hydroxylation activity were 6.5 and 30 °C, respectively. The and values of PH1 were 1.07 mM and 0.54 s, respectively, for this reaction by which 120 mM l-proline was converted to -4-hydroxy-l-proline with 92.8% yield (3.93 g·L·h). EDTA, [1,10-phenanthroline], Cu, Zn, Co, and Ni inhibited this reaction. PH1 was also active toward l-isoleucine for 4-hydroxyisoleucine synthesis. Additionally, the unique biophysical features of PH1 were predicted by molecular modeling whereby this study also contributes to our understanding of the catalytic mechanisms of 2-oxoglutarate-dependent dioxygenases.
羟基氨基酸在食品和制药行业具有巨大的潜在应用价值。然而，现有的双加氧酶在对游离氨基酸进行选择性高效羟基化方面存在局限性。在此，我们通过基因挖掘从[具体来源未给出]中鉴定出一种依赖于2-氧代戊二酸的双加氧酶，并对其编码蛋白（PH1）进行了表征。PH1的分子量估计为29 kDa。其L-脯氨酸羟基化活性的最适pH和温度分别为6.5和30℃。对于将120 mM L-脯氨酸转化为-4-羟基-L-脯氨酸且产率为92.8%（3.93 g·L·h）的该反应，PH1的Km和kcat值分别为1.07 mM和0.54 s-1。EDTA、[1,10-菲咯啉]、Cu、Zn、Co和Ni抑制该反应。PH1对L-异亮氨酸合成4-羟基异亮氨酸也具有活性。此外，通过分子建模预测了PH1独特的生物物理特征，该研究也有助于我们理解依赖于2-氧代戊二酸的双加氧酶的催化机制。
