{"doi":"10.1128/aac.00675-25","title":"Proposal of metagenomic-origin LRA-5 as a precursor of active β-lactamases through Tyr69Gln and Val166Glu amino acid substitutions: a functional and structural analysis","abstract":"ABSTRACT Wild-type LRA-5, recovered from Alaskan soil samples, shares no more than 33% amino acid sequence identity with enzymes from pathogens like PER β-lactamases. Recombinant E. coli expressing wild-type LRA-5 and its engineered variants LRA-5 Y69Q and LRA-5 V166E showed MIC values equivalent to control strains. However, LRA-5 Y69Q/V166E displayed MICs above the resistant breakpoint for some β-lactams. Kinetic parameters correlated with the MICs, showing that the catalytic efficiency of LRA-5 Y69Q/V166E was comparable to those from class A β-lactamases, such as CTX-M-15, PER-2, and KPC-2. LRA-5 Y69Q/V166E exhibited k cat / K m values up to 11,000-fold higher compared to wild-type LRA-5, which is associated with the presence of Glu166. The X-ray crystallographic structure of wild-type LRA-5 (1.80 Å; PDB 8EO5) shows that the lack of both Glu166 and a deacylation water molecule contributes to a biologically insignificant activity. Interactions observed between LRA-5 and ceftazidime (2.35 Å; PDB 8EO6) show structural conservation with other β-lactamases. In contrast, the crystallographic structure of LRA-5 Y69Q/V166E (2.15 Å; PDB 8EO7) bears a deacylation water molecule that is associated with the increase in catalytic activity compared to the wild-type variant. Circular dichroism results confirm that amino acid substitutions in LRA-5 do not affect the overall content of the secondary/tertiary structures. Evidence suggests that alternative evolutionary paths could have occurred for β-lactamases like LRA-5, produced by environmental microorganisms: (i) proteins having similar structural features than active β-lactamases may accumulate a small number of mutations (e.g., Y69Q/V166E) to yield active enzymes and (ii) the β-lactamase fold may have lost key residues in the absence of antibiotics.","journal":"Antimicrobial Agents and Chemotherapy","year":2025,"id":583532,"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":0.95,"is_data_producer":true,"deposit_databanks":{"PDB":["8EO7","8EO6","8EO5; orange","4D2O; blue","6CYK; yellow","CTX-M-14","8EU7"]},"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":776241,"name":"M.M. Rodríguez","orcid":"0000-0001-7507-5443","position":1,"is_corresponding":false},{"id":1302070,"name":"Pedro Penzotti","orcid":null,"position":2,"is_corresponding":false},{"id":776749,"name":"Florencia Brunetti","orcid":null,"position":3,"is_corresponding":false},{"id":776240,"name":"Bárbara Ghiglione","orcid":"0000-0002-1723-5135","position":4,"is_corresponding":false},{"id":1495682,"name":"Luke A. Moe","orcid":"0000-0001-6864-4660","position":5,"is_corresponding":false},{"id":451841,"name":"Daniela Centrón","orcid":"0000-0001-9308-3762","position":6,"is_corresponding":false},{"id":776243,"name":"Gabriel Gutkind","orcid":"0000-0003-1999-9785","position":7,"is_corresponding":false},{"id":1495683,"name":"Lin Gao","orcid":"0000-0002-7016-4612","position":8,"is_corresponding":false},{"id":333811,"name":"Shozeb Haider","orcid":"0000-0003-2650-2925","position":9,"is_corresponding":false},{"id":947199,"name":"R.A. Powers","orcid":"0000-0002-9968-8284","position":10,"is_corresponding":false},{"id":746387,"name":"Sebastián Klinke","orcid":"0000-0002-8777-0870","position":11,"is_corresponding":false},{"id":776244,"name":"P.P. Power","orcid":"0000-0002-7051-9954","position":12,"is_corresponding":false},{"id":1496107,"name":"Gabriela D'Amico González","orcid":null,"position":0,"is_corresponding":true}],"reference_count":61,"raw_metadata":null,"created_at":"2026-07-19T02:59:03.725721Z","pmid":"41312992","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":[]}