Unveiling 14 novel 2-hydroxy acid racemization and epimerization reactions in the lactate racemase superfamily.

2-hydroxy acids are organic carboxylic acids ubiquitous in the living world and are important building blocks in organic synthesis. Recently, the lactate racemase (LarA) superfamily, a diverse superfamily of 2-hydroxy acid racemases and epimerases using the nickel-pincer nucleotide (NPN) cofactor, has been uncovered. In this study, we performed a taxonomic analysis of the LarA superfamily, showing the distribution of lactate racemase homologs (LarAHs) sequences across the three domains of life. Subsequently, we overexpressed and purified nine LarAHs and investigated their biochemical properties and substrate specificities. We show that LarAHs from the lactate racemases group are more promiscuous than previously thought, with some members showing high specificity towards glycerate or 2-hydroxybutyrate. In other phylogenetic groups, we identified a new malate racemase and 2-hydroxyglutarate racemase, as well as a new 2-gluconate epimerase from an eukaryotic organism. We show that some LarAHs are able to isomerize up to 16 different substrates, mostly 2-hydroxy acids with hydrophobic side chains, thereby identifying 14 novel 2-hydroxy acid racemization and epimerization reactions catalyzed by LarAHs. These include the racemization of glycerate, 2-hydroxybutyrate, 2,4-dihydroxybutyrate, 2-hydroxyvalerate, 2-hydroxycaproate, 2,3-dihydroxyisovalérate, 2-hydroxy-3,3-dimethylbutyrate, 3-(4-hydroxyphenyl)lactate, 2-hydroxy-4-phenylbutyrate, and 2-hydroxy-4-oxo-4-phenylbutyrate. Additionally, we observed the C2-epimerization of all 2,3-dihydroxybutyrate stereoisomers (4-deoxy-DL-threonate and 4-deoxy-DL-erythronate) and the C2-epimerization of D-arabinarate epimers. Finally, through comparative analysis of Alphafold structural predictions, we identified key residues likely involved in substrate specificity and predicted the function of half of the LarAHs from the LarA superfamily. In conclusion, this study widely expands the scope of substrates isomerized by NPN-dependent enzymes.