{"doi":"10.1101/2022.11.09.515893","title":"Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor","abstract":"Abstract The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from Lactiplantibacillus plantarum (LarA Lp ) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarA Lp , but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH 4 ) irreversibly inactivates LarA Lp accompanied by a rapid color change of the enzyme. The drastically altered ultraviolet-visible spectra during NaBH 4 titration supported hydride transfer to C4 of NPN, and the concomitant Ni loss unraveled by mass spectrometry experiments accounted for the mechanism-based inactivation. High resolution structures of LarA Lp revealed a substantially weakened C-Ni bond in the metastable sulfite-NPN adduct where the NPN cofactor is in the reduced state. These findings allowed us to propose a mechanism of LarA Lp inactivation by NaBH 4 that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":313054,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9605,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":335784,"name":"Dexin Sui","orcid":null,"position":1,"is_corresponding":false},{"id":475596,"name":"Robert P. Hausinger","orcid":"0000-0002-3643-2054","position":2,"is_corresponding":false},{"id":334698,"name":"Jian Hu","orcid":"0000-0001-6657-9826","position":3,"is_corresponding":false},{"id":1010880,"name":"Santhosh Gatreddi","orcid":"0000-0003-0862-614X","position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":null,"created_at":"2026-07-19T00:33:40.460845Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}