Hydroxy acids (HAs) are organic carboxylic acids omnipresent in the living world, and are essential to various industries, including food, cosmetics, and pharmaceuticals. Recently, a superfamily of nickel-dependent HA racemases and epimerases using the nickel-pincer nucleotide (NPN) cofactor was discovered: the LarA superfamily. These enzymes, known as LarA homologs (LarAHs), are found in all domains of life and show potential for stereoselective conversion of chiral HAs. However, research on the LarA superfamily is still in its infancy, and much remains to be understood about the reactions catalyzed by LarAHs. This thesis delves into the exploration and biotechnological potential of the LarA superfamily through biochemical characterization, taxonomic analysis, and structural modeling. We developed a versatile capillary electrophoresis (CE) method for efficient chiral analysis of underivatized α-, β- and poly-hydroxy acids (AHAs, BHAs, PHAs). Using this CE method, we explored and expanded five different LarAH families within the LarA superfamily: lactate racemases (LARs), short-chain aliphatic HA racemases (SARs), malate racemases (MARs), α-hydroxyglutarate racemases (HGRs), and D-gluconate 2-epimerase (GntEs). We also identified two novel families of LarAHs: hydrophobic HA racemases (HHRs), and broad-spectrum HA isomerases (BSHIs). In total, we characterized 13 LarAHs, revealing 11 novel AHA racemization reactions and 23 novel PHA/sugar acid C2-epimerization reactions. Altogether, these findings significantly broaden the substrate scope of NPN-dependent enzymes from the LarA superfamily, offering new possibilities for producing valuable HA stereoisomers and paving the way for future biotechnological applications of LarAHs. (SC - Sciences) -- UCL, 2024