biostudies-literatureKawai SJapan Society for the Promotion of ScienceNew Energy and Industrial Technology Development Organizatione202404790https://www.ebi.ac.uk/biostudies/studies/S-EPMC11973848biostudies-literatureUnknown31(20)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 (2SChemistry (Weinheim an der Bergstrasse, Germany)Identification of Two Distinct Stereoselective Lysine 5-Hydroxylases by Genome Mining Based on Alazopeptin Biosynthetic Enzymes.PMC1197384819H0464522KJ104619H0568522H05130Moriga KNirdnoy WOgawa JOhnishi YHara RKawai SKatsuyama YfalseIdentification 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/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 (2S2025-01-01T00:00:00Z2025 Apr2025-07-14T03:02:48.936Z2025-07-14T03:02:48.936ZS-EPMC119738483996043610.1002/chem.202404790