Highly Regioselective and Stereoselective Hydroxylation of Free Amino Acids by a 2-Oxoglutarate-Dependent Dioxygenase from Kutzneria albida

[Image: see text] 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 Kutzneria albida by gene mining and characterized the encoded protein (KaPH1). KaPH1 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 K(m) and k(cat) values of KaPH1 were 1.07 mM and 0.54 s(–1), respectively, for this reaction by which 120 mM l-proline was converted to trans-4-hydroxy-l-proline with 92.8% yield (3.93 g·L(–1)·h(–1)). EDTA, [1,10-phenanthroline], Cu(2+), Zn(2+), Co(2+), and Ni(2+) inhibited this reaction. KaPH1 was also active toward l-isoleucine for 4-hydroxyisoleucine synthesis. Additionally, the unique biophysical features of KaPH1 were predicted by molecular modeling whereby this study also contributes to our understanding of the catalytic mechanisms of 2-oxoglutarate-dependent dioxygenases.