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

Abstract

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.