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

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/Fe²⁺-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.