{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T11:08:33Z","timestamp":1792062513858,"version":"4.4.0"},"reference-count":81,"publisher":"Wiley","issue":"17","license":[{"start":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T00:00:00Z","timestamp":1740441600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T00:00:00Z","timestamp":1740441600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100010663","name":"H2020 European Research Council","doi-asserted-by":"publisher","award":["885396"],"award-info":[{"award-number":["885396"]}],"id":[{"id":"10.13039\/100010663","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010665","name":"H2020 Marie Sk\u0142odowska-Curie Actions","doi-asserted-by":"publisher","award":["HORIZON-MSCA-2022-PF-01-101106886"],"award-info":[{"award-number":["HORIZON-MSCA-2022-PF-01-101106886"]}],"id":[{"id":"10.13039\/100010665","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001671","name":"European Synchrotron Radiation Facility","doi-asserted-by":"publisher","award":["MX-2526"],"award-info":[{"award-number":["MX-2526"]}],"id":[{"id":"10.13039\/501100001671","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Angew Chem Int Ed"],"published-print":{"date-parts":[[2025,4,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Artificial metalloenzymes (ArMs) are an attractive approach to achieving \u201cnew to nature\u201d biocatalytic transformations. In this work, a novel copper\u2010dependent artificial Michaelase (Cu_Michaelase) comprising a genetically encoded copper\u2010binding ligand, i.\u2009e. (2,2\u2010bipyridin\u20105\u2010yl)alanine (BpyA), was developed. For the first time, such an ArM containing a non\u2010canonical metal\u2010binding amino acid was successfully optimized through directed evolution. The evolved Cu_Michaelase was applied in the copper\u2010catalyzed asymmetric addition of 2\u2010acetyl azaarenes to nitroalkenes, yielding various \u03b3\u2010nitro butyric acid derivatives, which are precursors for a range of high\u2010value\u2010added pharmaceutically relevant compounds, with good yields and high enantioselectivities (up to &gt;99\u2009% yield and 99\u2009%\u2005ee). Additionally, the evolved variant could be further used in a preparative\u2010scale synthesis, providing chiral products for diverse derivatizations. X\u2010ray crystal structure analysis confirmed the binding of Cu(II) ions to the BpyA residues and showed that, in principle, there is sufficient space for the 2\u2010acetyl azaarene substrate to coordinate. Kinetic studies showed that the increased catalytic efficiency of the evolved enzyme is due to improvements in apparent\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>M<\/jats:sub>\n                    for both substrates and a notable threefold increase in apparent\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>cat<\/jats:sub>\n                    for 2\u2010acetyl pyridine. This work illustrates the potential of artificial metalloenzymes exploiting non\u2010canonical metal\u2010binding ligands for new\u2010to\u2010nature biocatalysis.\n                  <\/jats:p>","DOI":"10.1002\/anie.202423182","type":"journal-article","created":{"date-parts":[[2025,2,13]],"date-time":"2025-02-13T09:01:27Z","timestamp":1739437287000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["An Artificial Copper\u2010Michaelase Featuring a Genetically Encoded Bipyridine Ligand for Asymmetric Additions to Nitroalkenes"],"prefix":"10.1002","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3825-0534","authenticated-orcid":false,"given":"Ru","family":"Jiang","sequence":"first","affiliation":[{"name":"Stratingh Institute for Chemistry University of Groningen  Nijenborgh 3 9747 AG Groningen The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-2699-5439","authenticated-orcid":false,"given":"Fabrizio","family":"Casilli","sequence":"additional","affiliation":[{"name":"Stratingh Institute for Chemistry University of Groningen  Nijenborgh 3 9747 AG Groningen The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1915-9850","authenticated-orcid":false,"given":"Andy\u2010Mark W. H.","family":"Thunnissen","sequence":"additional","affiliation":[{"name":"Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen  Nijenborgh 3 4 9747 AG Groningen The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0364-9564","authenticated-orcid":false,"given":"Gerard","family":"Roelfes","sequence":"additional","affiliation":[{"name":"Stratingh Institute for Chemistry University of Groningen  Nijenborgh 3 9747 AG Groningen The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,2,25]]},"reference":[{"key":"e_1_2_8_1_1","unstructured":"Reviews of Artificial Metalloenzyme:"},{"key":"e_1_2_8_2_2","doi-asserted-by":"publisher","DOI":"10.1021\/cr400458x"},{"key":"e_1_2_8_3_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.7b00014"},{"key":"e_1_2_8_4_2","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.7b03754"},{"key":"e_1_2_8_5_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.9b00004"},{"key":"e_1_2_8_6_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.8b00586"},{"key":"e_1_2_8_7_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.1c00829"},{"key":"e_1_2_8_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2024.102508"},{"key":"e_1_2_8_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jinorgbio.2024.112621"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1060077"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.052308.105824"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-019-0410-8"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.4c00120"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.4c00136"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1039\/C4SC01525H"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1039\/C7SC03477F"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1039\/C7OB00390K"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1039\/D3CY01648J"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202000306"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.9b03739"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.2165\/11319230-000000000-00000"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsomega.1c05378"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature14343"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules27123797"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.8b04299"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1021\/ol060576e"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tetasy.2014.02.001"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.7b00692"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/1099-0690(200206)2002:12<1877::AID-EJOC1877>3.0.CO;2-U"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.2174\/138527212803520128"},{"key":"e_1_2_8_31_1","unstructured":"\u00a0"},{"key":"e_1_2_8_32_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201107404"},{"key":"e_1_2_8_33_2","doi-asserted-by":"publisher","DOI":"10.1002\/chem.201302351"},{"key":"e_1_2_8_34_2","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201200676"},{"key":"e_1_2_8_35_2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms10911"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.6b11928"},{"key":"e_1_2_8_37_1","author":"Zhu Z.","year":"2024","journal-title":"Angew. Chem. Int. Ed."},{"key":"e_1_2_8_38_1","unstructured":"For examples of metal catalyzed conjugate addition of 2-acetyl azaarenes to nitroalkenes.Nickle-catalyzed:"},{"key":"e_1_2_8_39_2","doi-asserted-by":"publisher","DOI":"10.1021\/ol400578c"},{"key":"e_1_2_8_40_2","doi-asserted-by":"publisher","DOI":"10.1039\/C5CC08480F"},{"key":"e_1_2_8_41_2","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.3c12555"},{"key":"e_1_2_8_42_2","doi-asserted-by":"publisher","DOI":"10.1039\/C6QO00562D"},{"key":"e_1_2_8_43_2","doi-asserted-by":"publisher","DOI":"10.1002\/adsc.201701377"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1055\/s-2007-992386"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01151-07"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1038\/emboj.2008.263"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.200703397"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja808340b"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.5b05790"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-019-0420-6"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.1c02298"},{"key":"e_1_2_8_52_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41557-018-0082-z"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.1c00996"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07391-3"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja500613n"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2016.019"},{"key":"e_1_2_8_57_1","unstructured":"Deposition numbers 9H9\u2005W (for LmrR_V15BpyA_KK in an open state) 9H9X (for LmrR_V15BpyA_KK in a closed state) 9H9Y (for LmrR_BVS_KK) 9H9Z (for LmrR_V15BpyA_KK_Cu) 9HA0 (for LmrR_BVS _KK_Cu) contain the supplementary crystallographic data for this paper. These data are provided free of charge by the Worldwide Protein Data Bank.."},{"key":"e_1_2_8_58_1","unstructured":"For crystallization the proteins with two lysines (K55 and K59) in the DNA binding domain were used as their presence generally improves crystallization; For catalysis mutated proteins (K55D_K59Q) were used to facilitate expression and purification."},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1002\/ejoc.201201579"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.3390\/catal11091134"},{"key":"e_1_2_8_61_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202000129"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.6023\/cjoc201709052"},{"key":"e_1_2_8_63_1","unstructured":"MagStrep\u00ae Strep-Tactin\u00aeXT bead-based protein purification:https:\/\/www.iba-lifesciences.com\/magstrep-strep-tactin-xt-beads\/2\u20135090-002."},{"key":"e_1_2_8_64_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444913015308"},{"key":"e_1_2_8_65_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444909047337"},{"key":"e_1_2_8_66_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444913000061"},{"key":"e_1_2_8_67_1","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798323003595"},{"key":"e_1_2_8_68_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0021889807021206"},{"key":"e_1_2_8_69_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201813499"},{"key":"e_1_2_8_70_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"e_1_2_8_71_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444911001314"},{"key":"e_1_2_8_72_1","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798317000067"},{"key":"e_1_2_8_73_1","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444912001308"},{"key":"e_1_2_8_74_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3330"},{"key":"e_1_2_8_75_1","doi-asserted-by":"publisher","DOI":"10.1016\/0378-1119(90)90336-P"},{"key":"e_1_2_8_76_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201301123"},{"key":"e_1_2_8_77_1","doi-asserted-by":"publisher","DOI":"10.1055\/s-2007-992386"},{"key":"e_1_2_8_78_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.orglett.0c03100"},{"key":"e_1_2_8_79_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.201405847"},{"key":"e_1_2_8_80_1","doi-asserted-by":"publisher","DOI":"10.1021\/ol3002514"},{"key":"e_1_2_8_81_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.orglett.9b04263"}],"container-title":["Angewandte Chemie International Edition"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/anie.202423182","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/anie.202423182","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/anie.202423182","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T14:16:36Z","timestamp":1775916996000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/anie.202423182"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,25]]},"references-count":81,"journal-issue":{"issue":"17","published-print":{"date-parts":[[2025,4,17]]}},"alternative-id":["10.1002\/anie.202423182"],"URL":"https:\/\/doi.org\/10.1002\/anie.202423182","archive":["Portico"],"relation":{},"ISSN":["1433-7851","1521-3773"],"issn-type":[{"value":"1433-7851","type":"print"},{"value":"1521-3773","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,25]]},"assertion":[{"value":"2024-11-27","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-02-25","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e202423182"}}