Identification of Two Distinct Stereoselective Lysine 5-Hydroxylases by Genome Mining Based on Alazopeptin Biosynthetic Enzymes

Enzymes that catalyze regioselective and stereoselective hydroxylation of amino acids are useful tools for the synthesis of pharmaceuticals. AzpK is an unprecedented lysine 5-hydroxylase that is involved in alazopeptin biosynthesis, although its enzymatic activity has not been confirmed in vitro. Here, we identified two α-ketoglutarate/Fe-dependent dioxygenases in Actinosynnema mirum and Pseudomonas psychrotolerans (Am_AzpK2 and Pp_AzpK2, respectively) as lysine 5-hydroxylases, using genome mining based on the alazopeptin biosynthetic gene cluster. Interestingly, Am_AzpK2 and Pp_AzpK2 synthesized different isomers, (2S,5S)- and (2S,5R)-5-hydroxylysine, respectively. We also identified two AzpJ homologs as the dehydrogenases that specifically recognize the hydroxy groups of (2S,5S)- and (2S,5R)-5-hydroxylysine to synthesize a keto group. These dehydrogenases were shown to be useful tools for characterizing the stereochemistry of 5-hydroxylysine and evaluating the activity of lysine 5-hydroxylases. Furthermore, we identified three lysine 5-hydroxylases that synthesize (2S,5S)-5-hydroxylysine and four lysine 5-hydroxylases that synthesize (2S,5R)-5-hydroxylysine from the genome database. Genome scanning based on lysine 5-hydroxylases indicated the presence of undiscovered natural products with 5-hydroxylysine moieties. In conclusion, this study provides a fundamental technology for the stereoselective production of 5-hydroxylysine. Further analysis of the stereoselective lysine 5-hydroxylases would reveal how nature establishes highly stereoselective hydroxylation.