Iron(II)/α-ketoacid-dependent oxygenases (αKAOs) are enzymes that catalyze the oxidation of unactivated C-H bonds, mainly through hydroxylation. Among these, those that are active towards amino-acids and their derivatives are grouped in the Clavaminate Synthase Like (CSL) family. CSL enzymes exhibit high regio-and stereoselectivities with strict substrate specificity. This study reports the structural elucidation of two new regiodivergent members, KDO1 and KDO5, active towards lysine, and the structural and computational analysis of the whole family through modelling and classification of active sites. The structures of KDO1 and KDO5 in complex with their ligands show that one exact position in the active site controls the regioselectivity of the reaction. Our results suggest that the substrate specificity and high stereoselectivity typical of this family is linked to a lid that closes up in order to form a sub-pocket around the side chain of the substrate. This dynamic lid is found throughout the family with varying sequence and length and is associated with a conserved stable dimeric interface. Results from this study could be a starting-point for exploring the functional diversity of the CSL family and direct in vitro screening in the search for new enzymatic activities. Enzymes derived from microorganisms are particularly appealing for the design of chemical processes that are environmentally friendly and sustainable 1. Enzymes catalyze interconversions of many functional groups with well-defined chemo-, regio-and stereoselectivities, resulting in high atom economy and reduced waste during synthesis. Over the past 25 years, the tremendous progress in molecular biology, bioinformatics and process optimization have lowered the biotechnological barriers that were limiting the industrial use of enzymes 2-4. Much research has looked for novel enzymes able to catalyze challenging reactions such as the functionalization of non-activated (i.e. not alpha to CC or C-heteroatom double bonds) C-H bonds. Such reactions still resist most efforts made by organic chemists. However, enzymes that can catalyze the formation of various C-heteroatom bonds have been found 5,6. We have focused our work on the iron(II)/α-KetoAcid-dependent Oxygenases (αKAOs), a mechanistically diverse superfamily of enzymes that use dioxygen as the oxidant to catalyze several reactions via C-H bond activation, mostly hydroxylation 7-9. These enzymes operate with three substrates, diox-ygen, α-ketoglutarate (α-KG) and a primary substrate. One oxygen atom is transferred to a primary substrate to give an oxidized product, and the second oxygen is given to the α-KG, which upon oxidation, forms succinate and CO 2 10,11,12. Many αKAOs are tailoring enzymes involved in secondary metabolite biosynthesis. Such enzymes, often found in bacteria, have been reported to hydroxylate the side chains of free amino acids and derivatives, or tether peptides in non-ribosomal peptide biosynthesis 13. Since hydroxylated amino acids contain several stereogenic centers, they are valuable chiral building blocks for fine chemical synthesis, which justifies the intensive effort