{"doi":"10.1016/j.jbc.2025.108382","title":"Substrate recognition in Bacillus anthracis sortase B beyond its canonical pentapeptide binding motif and use in sortase-mediated ligation","abstract":"Sortases are critical cysteine transpeptidases that facilitate the attachment of proteins to the cell wall in Gram-positive bacteria. These enzymes are potential targets for novel antibiotic development, and versatile tools in protein engineering applications. There are six classes of sortases recognized, yet class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes endogenously recognize the amino acid sequence LPXTG, where X = any amino acid, with additional promiscuity now recognized in multiple positions for certain SrtA enzymes. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes were previously shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. Finally, we use chimeric proteins to identify mutations that improve baSrtB activity by ∼4-fold, and demonstrate the feasibility of sortase-mediated ligation using a baSrtB enzyme variant. These studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.","journal":"Journal of Biological Chemistry","year":2025,"id":543300,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9614,"is_data_producer":false,"deposit_databanks":null,"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":897434,"name":"Darren E. Lee","orcid":"0000-0002-5053-863X","position":1,"is_corresponding":false},{"id":1118476,"name":"Jadon M. Blount","orcid":null,"position":2,"is_corresponding":false},{"id":1433373,"name":"Kayla A. Croney","orcid":null,"position":3,"is_corresponding":false},{"id":1433374,"name":"Justin W. Ibershof","orcid":null,"position":4,"is_corresponding":false},{"id":1433375,"name":"Caroline Ceravolo","orcid":null,"position":5,"is_corresponding":false},{"id":1433376,"name":"Kate M. Brown","orcid":null,"position":6,"is_corresponding":false},{"id":1120292,"name":"Noah J. Goodwin-Rice","orcid":null,"position":7,"is_corresponding":false},{"id":1433377,"name":"Kyle M. Whitham","orcid":null,"position":8,"is_corresponding":false},{"id":274303,"name":"James McCarty","orcid":"0000-0002-3838-6004","position":9,"is_corresponding":false},{"id":720452,"name":"John M. Antos","orcid":"0000-0002-2555-1875","position":10,"is_corresponding":false},{"id":315621,"name":"Jeanine F. Amacher","orcid":"0000-0002-3646-7521","position":11,"is_corresponding":false},{"id":897433,"name":"Sophie N. Jackson","orcid":"0000-0001-9133-6906","position":0,"is_corresponding":true}],"reference_count":82,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:53:04.088079Z","pmid":"40049417","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":[]}