biostudies-literatureUnknownGatreddi SNIGMS NIH HHSThe nickel pincer nucleotide (NPN) cofactor catalyzes the racemization/epimerization of α-hydroxy acids in enzymes of the LarA family. The established proton-coupled hydride transfer mechanism requires two catalytic histidine residues that alternately act as general acids and general bases. Notably, however, a fraction of LarA homologs (LarAHs) lack one of the active site histidine residues, replacing it with an asparaginyl side chain that cannot participate in acid/base catalysis. Here, we investigated two such LarAHs and solved their cryo-electron microscopic structures with and without loaded NPN cofactor, respectively. The structures revealed a consistent octameric assembly that is unprecedented in the LarA family and unveiled a new set of active site residues that likely recognize andbioRxiv : the preprint server for biology2025.08.19.671153https://www.ebi.ac.uk/biostudies/studies/S-EPMC12393405biostudies-literatureStructures of two LarA-like nickel-pincer nucleotide cofactor-utilizing enzymes with a single catalytic histidine residue.PMC12393405R35 GM140931R01 GM128959R35 GM140803Desguin BSui DHu JWang TUrdiain-Arraiza JHausinger RPGatreddi SParent KNSubramanian SfalseStructures of two LarA-like nickel-pincer nucleotide cofactor-utilizing enzymes with a single catalytic histidine residue.The nickel pincer nucleotide (NPN) cofactor catalyzes the racemization/epimerization of α-hydroxy acids in enzymes of the LarA family. The established proton-coupled hydride transfer mechanism requires two catalytic histidine residues that alternately act as general acids and general bases. Notably, however, a fraction of LarA homologs (LarAHs) lack one of the active site histidine residues, replacing it with an asparaginyl side chain that cannot participate in acid/base catalysis. Here, we investigated two such LarAHs and solved their cryo-electron microscopic structures with and without loaded NPN cofactor, respectively. The structures revealed a consistent octameric assembly that is unprecedented in the LarA family and unveiled a new set of active site residues that likely recognize and2025-01-01T00:00:00Z2025 Aug2026-05-29T03:08:37.631Z2026-05-29T03:05:38.807ZS-EPMC123934054089470010.1101/2025.08.19.671153