biostudies-literatureGatreddi SNIGMS NIH HHS2857-2866https://www.ebi.ac.uk/biostudies/studies/S-EPMC11851776biostudies-literatureUnknown15(4)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 solveACS catalysisStructural Basis for the Catalysis and Substrate Specificity of a LarA Racemase with a Broad Substrate Spectrum.PMC11851776R35 GM140931R01 GM128959Desguin BHu JUrdiain-Arraiza JHausinger RPGatreddi SfalseStructural 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 solve2025-01-01T00:00:00Z2025 Feb2026-06-02T19:15:33.099Z2025-04-04T02:47:48.048ZS-EPMC118517764001325010.1021/acscatal.4c07804