{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T01:36:15Z","timestamp":1777080975702,"version":"3.51.4"},"reference-count":67,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:00:00Z","timestamp":1760140800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["184.035.002"],"award-info":[{"award-number":["184.035.002"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["ECHO 711.016.002"],"award-info":[{"award-number":["ECHO 711.016.002"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003246","name":"Nederlandse Organisatie voor Wetenschappelijk Onderzoek","doi-asserted-by":"publisher","award":["OCENW.KLEIN.264"],"award-info":[{"award-number":["OCENW.KLEIN.264"]}],"id":[{"id":"10.13039\/501100003246","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2025,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Epistasis, the non\u2010additive effect of mutations, substantially undermines our ability to predict the evolutionary trajectories of enzymes. Epistatic effects are evident in the evolution of serine \u03b2\u2010lactamases, where synergistic mutations can enhance antimicrobial resistance. We recently demonstrated that positive epistasis drives clavulanic acid resistance in double\u2010mutant libraries of the \u03b2\u2010lactamase BlaC. Here, we employed various biochemical and structural approaches to investigate molecular mechanisms underlying epistasis in the fitness of two double mutant variants, I105Y\u2010S130G and I105G\u2010G132N. For the latter enzyme, epistatic compensation of catalytic activity was detected for multiple substrates and proved to be highly buffer\u2010dependent. Non\u2010additive effects were also evident in the thermostability profile of the I105G\u2010G132N variant. The interplay between the reduced clavulanic acid sensitivity of the S130G and G132N variants and active\u2010site modifications induced by Ile105 substitutions is discussed. The results demonstrate that the origins of epistasis can be rooted in multiple enzyme traits, highlighting its important role in the evolution of antimicrobial resistance.<\/jats:p>","DOI":"10.1002\/pro.70325","type":"journal-article","created":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:44:06Z","timestamp":1760190246000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Phosphate ions modulate enzyme activity and epistatic effects in two clavulanic acid\u2010resistant \u03b2\u2010lactamase mutants"],"prefix":"10.1002","volume":"34","author":[{"given":"Marko","family":"Radojkovi\u0107","sequence":"first","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saar F.","family":"Koene","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra","family":"Chikunova","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bogdan I.","family":"Florea","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sivanandam V.","family":"Natarajan","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aimee L.","family":"Boyle","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"},{"name":"School of Chemistry University of Bristol  Bristol UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2615-6914","authenticated-orcid":false,"given":"Marcellus","family":"Ubbink","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry Leiden University  Leiden The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,10,11]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1042\/bj2760269"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.97.7.3160"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0112456"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-022-00856-6"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature11510"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-44333-5"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2017.09.005"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi002277h"},{"key":"e_1_2_8_10_1","first-page":"1","article-title":"Cryptic phenotypic variation emerges rapidly during the adaptive evolution of a carbapenemase","volume":"7","author":"Dabos L","year":"2025","journal-title":"Nat Ecol Evol"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja027704o"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M407606200"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2617"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.7b00556"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1107\/S090744491003982X"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0073123"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-024-01117-4"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msw052"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2019.03.020"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-07649-1"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M306059200"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi701506h"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijantimicag.2006.09.005"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2400439121"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1107\/S2052252514009324"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-023-04422-z"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2313513121"},{"key":"e_1_2_8_28_1","first-page":"1040","article-title":"Enzyme efficiency but not thermostability drives cefotaxime resistance evolution in TEM\u20101 \u03b2\u2010lactamase","volume":"34","author":"Knies JL","year":"2017","journal-title":"Mol Biol Evol"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.39.12.2593"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01253-13"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.3103\/S0027131410030077"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1371\/annotation\/d618ce28-5010-47df-a44e-148ecdc0fef6"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004949"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01990-20"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2021.04.007"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2876"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0039822"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1142819"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1098\/rspb.2011.0870"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.abd3623"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.454"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msu113"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.38.4.767"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2452"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.41.4.838"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2025.110347"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-024-05868-5"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2025.168999"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msac194"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/mst096"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2011.10.041"},{"key":"e_1_2_8_52_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2020.08.005"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00598-15"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2897"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2016.04.033"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1038\/359700a0"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2023.126160"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.1128\/aac.01510-23"},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0502700"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2009.11.005"},{"key":"e_1_2_8_61_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi8001055"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.1111\/febs.16892"},{"key":"e_1_2_8_63_1","doi-asserted-by":"publisher","DOI":"10.1002\/prot.20449"},{"key":"e_1_2_8_64_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00320-06"},{"key":"e_1_2_8_65_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1123539"},{"key":"e_1_2_8_66_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0101813"},{"key":"e_1_2_8_67_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910045749"},{"key":"e_1_2_8_68_1","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20120136"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.70325","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T13:02:17Z","timestamp":1763125337000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.70325"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,11]]},"references-count":67,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2025,11]]}},"alternative-id":["10.1002\/pro.70325"],"URL":"https:\/\/doi.org\/10.1002\/pro.70325","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"value":"0961-8368","type":"print"},{"value":"1469-896X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,11]]},"assertion":[{"value":"2025-05-12","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-09-23","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-10-11","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70325"}}