Structural Basis for the Catalysis and Substrate Specificity of a LarA Racemase with a Broad Substrate Spectrum

The LarA family consists of diverse racemases/epimerases that interconvert the diastereomers of α-hydroxyacids by using a nickel-pincer nucleotide (NPN) cofactor. The hidden redox reaction catalyzed by the NPN cofactor makes LarA enzymes attractive engineering targets for various applications. However, how a LarA enzyme binds its natural substrate and recognizes different α-hydroxyacids has not been elucidated. Here, we report three high-resolution structures of the enzyme-substrate complexes of a broad-spectrum LarA enzyme from <i>Isosphaera pallida</i> (LarA <i>_Ip</i> ). The substrate binding mode reveals a near-optimal orientation and distance between the hydride donor and acceptor, consistent with an updated proton-coupled hydride transfer mechanism. The experimentally solved structures, together with the structural models of other LarA enzymes, lead to the identification of the residues/structural elements that are critically involved in the interactions with different α-hydroxyacids. Collectively, this work provides a structural basis for the catalysis and substrate specificity of the LarA enzymes.