{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,12]],"date-time":"2026-03-12T05:05:19Z","timestamp":1773291919949,"version":"3.50.1"},"reference-count":30,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2014,12,26]],"date-time":"2014-12-26T00:00:00Z","timestamp":1419552000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"German Academic Exchange Service (DAAD); NSERC-Vanier Scholar; Natural Sciences and Engineering Research Council of Canada"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2015,4]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid\u2010encoded enzyme that is intrinsically resistant to the widely\u2010administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to <jats:italic>E. coli<\/jats:italic> chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding\/catalytic event at any given time. As a result, substitution of an active\u2010site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such \u201chalf\u2010native\u201d tetramers were shown to procure native\u2010like catalytic activity, with similar <jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub> values but <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub> values 5\u2010 to 33\u2010fold lower, illustrating a high tolerance for active\u2010site substitutions. The selected variants showed a reduced thermal stability (<jats:italic>T<\/jats:italic><jats:sub>m<\/jats:sub> \u223c12\u00b0C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active\u2010site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native\u2010like catalytic rate and thus ensure cellular proliferation.<\/jats:p>","DOI":"10.1002\/pro.2602","type":"journal-article","created":{"date-parts":[[2014,11,17]],"date-time":"2014-11-17T14:48:00Z","timestamp":1416235680000},"page":"495-507","source":"Crossref","is-referenced-by-count":10,"title":["Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active\u2010site substitutions"],"prefix":"10.1002","volume":"24","author":[{"given":"Maximilian C. C. J. C.","family":"Ebert","sequence":"first","affiliation":[{"name":"D\u00e9partement de Biochimie Universit\u00e9 de Montr\u00e9al C.P. 6128, Succursale Centre\u2010Ville Montr\u00e9al Qu\u00e9bec H3C 3J7 Canada"},{"name":"PROTEO, the Qu\u00e9bec Network for Protein Function, Structure and Engineering  Qu\u00e9bec Canada"},{"name":"CGCC, the Center for Green Chemistry and Catalysis Montr\u00e9al Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Krista L.","family":"Morley","sequence":"additional","affiliation":[{"name":"PROTEO, the Qu\u00e9bec Network for Protein Function, Structure and Engineering  Qu\u00e9bec Canada"},{"name":"D\u00e9partement de Chimie Universit\u00e9 de Montr\u00e9al C.P. 6128, Succursale Centre\u2010Ville Montr\u00e9al Qu\u00e9bec H3C 3J7 Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jordan P.","family":"Volpato","sequence":"additional","affiliation":[{"name":"D\u00e9partement de Biochimie Universit\u00e9 de Montr\u00e9al C.P. 6128, Succursale Centre\u2010Ville Montr\u00e9al Qu\u00e9bec H3C 3J7 Canada"},{"name":"PROTEO, the Qu\u00e9bec Network for Protein Function, Structure and Engineering  Qu\u00e9bec Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andreea R.","family":"Schmitzer","sequence":"additional","affiliation":[{"name":"CGCC, the Center for Green Chemistry and Catalysis Montr\u00e9al Canada"},{"name":"D\u00e9partement de Chimie Universit\u00e9 de Montr\u00e9al C.P. 6128, Succursale Centre\u2010Ville Montr\u00e9al Qu\u00e9bec H3C 3J7 Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joelle N.","family":"Pelletier","sequence":"additional","affiliation":[{"name":"D\u00e9partement de Biochimie Universit\u00e9 de Montr\u00e9al C.P. 6128, Succursale Centre\u2010Ville Montr\u00e9al Qu\u00e9bec H3C 3J7 Canada"},{"name":"PROTEO, the Qu\u00e9bec Network for Protein Function, Structure and Engineering  Qu\u00e9bec Canada"},{"name":"CGCC, the Center for Green Chemistry and Catalysis Montr\u00e9al Canada"},{"name":"D\u00e9partement de Chimie Universit\u00e9 de Montr\u00e9al C.P. 6128, Succursale Centre\u2010Ville Montr\u00e9al Qu\u00e9bec H3C 3J7 Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2014,12,26]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-1605(00)00351-2"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00363a005"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/47.4.495"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.200400237"},{"key":"e_1_2_7_6_1","unstructured":".CCG. Inc. (2014) Molecular operating environment (MOE) 2013.08. 1010 Sherbooke St. West Suite #910 Montreal QC Canada H3A 2R7."},{"key":"e_1_2_7_7_1","doi-asserted-by":"crossref","first-page":"10857","DOI":"10.1016\/S0021-9258(19)86600-0","article-title":"The amino acid sequence of the trimethoprim\u2010resistant dihydrofolate reductase specified in Escherichia coli by R\u2010plasmid R67","volume":"254","author":"Stone D","year":"1979","journal-title":"J Biol Chem"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsb1195-1018"},{"key":"e_1_2_7_9_1","first-page":"1316","article-title":"Novel crystallization conditions for tandem variant R67 DHFR yield a wild\u2010type crystal structure","volume":"67","author":"Yachnin BJ","year":"2011","journal-title":"Acta Cryst"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0110544"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00058a002"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/10.12.1415"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi960205d"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzh090"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi049646k"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1432-1033.1976.tb10055.x"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M105107200"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi701532r"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi1005943"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi701455x"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1996.tb00461.x"},{"key":"e_1_2_7_22_1","doi-asserted-by":"crossref","first-page":"22672","DOI":"10.1016\/S0021-9258(18)41580-3","article-title":"Artificial duplication of the R67 dihydrofolate reductase gene to create protein asymmetry: effects on protein activity and folding","volume":"268","author":"Zhuang P","year":"1993","journal-title":"J Biol Chem"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.2000.4051"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1074-5521(98)90616-0"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.321"},{"key":"e_1_2_7_26_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0605224103"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1039\/c1cp21187k"},{"key":"e_1_2_7_28_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm201645r"},{"key":"e_1_2_7_29_1","doi-asserted-by":"publisher","DOI":"10.1038\/188231a0"},{"key":"e_1_2_7_30_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.4"},{"key":"e_1_2_7_31_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac60214a047"}],"container-title":["Protein Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fpro.2602","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fpro.2602","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/pro.2602","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,13]],"date-time":"2023-09-13T20:35:24Z","timestamp":1694637324000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/pro.2602"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,12,26]]},"references-count":30,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2015,4]]}},"alternative-id":["10.1002\/pro.2602"],"URL":"https:\/\/doi.org\/10.1002\/pro.2602","archive":["Portico"],"relation":{},"ISSN":["0961-8368","1469-896X"],"issn-type":[{"value":"0961-8368","type":"print"},{"value":"1469-896X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,12,26]]}}}