{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T03:27:39Z","timestamp":1782444459751,"version":"3.54.5"},"reference-count":90,"publisher":"Oxford University Press (OUP)","issue":"10","license":[{"start":{"date-parts":[[2025,9,16]],"date-time":"2025-09-16T00:00:00Z","timestamp":1757980800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000038","name":"NSERC","doi-asserted-by":"publisher","award":["RGPIN-N-2018-04686"],"award-info":[{"award-number":["RGPIN-N-2018-04686"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003977","name":"Israel Science Foundation","doi-asserted-by":"publisher","award":["1764\/21"],"award-info":[{"award-number":["1764\/21"]}],"id":[{"id":"10.13039\/501100003977","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001658","name":"the Minerva Foundation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001658","id-type":"DOI","asserted-by":"crossref"}]},{"name":"APRENTICE"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,10,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>How does enzymatic activity emerge? To shed light on this fundamental question, we study type B dihydrofolate reductases (DfrB), which were discovered for their role in antibiotic resistance. These rudimentary enzymes are evolutionarily distinct from the ubiquitous, monomeric FolA dihydrofolate reductases targeted by the antibiotic trimethoprim. DfrB is unique: it homotetramerizes to form a highly symmetrical central tunnel that accommodates its substrates in close proximity and the right orientation, thus promoting the metabolically essential production of tetrahydrofolate. It is the only known enzyme built from the ancient Src Homology 3 fold, typically a binding module. Strikingly, by studying the evolution of this enzyme family, we observe that no active-site residues are conserved across catalytically active homologs. Integrating experimental and computational analyses, we identify an intricate relationship between homotetramerization and catalytic activity, where formation of a tunnel featuring positive electrostatic potential proves to be a powerful predictor of activity. We demonstrate that the DfrB enzymes have not evolved in response to the synthetic antibiotic to which they confer strong resistance, and propose that DfrB domains evolved the capacity for rudimentary catalysis from a binding capacity. That (rudimentary) catalysis can emerge from the homotetramerization of a binding domain, and that it has been recently recruited by pathogenic bacteria, manifests the opportunistic nature of evolution.<\/jats:p>","DOI":"10.1093\/molbev\/msaf215","type":"journal-article","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T22:25:54Z","timestamp":1758320754000},"source":"Crossref","is-referenced-by-count":2,"title":["From Binding to Catalysis: Emergence of a Rudimentary Enzyme Conferring Intrinsic Antibiotic Resistance"],"prefix":"10.1093","volume":"42","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4865-0267","authenticated-orcid":false,"given":"Claud\u00e8le","family":"Lemay-St-Denis","sequence":"first","affiliation":[{"name":"PROTEO, The Qu\u00e9bec Network for Research on Protein, Function, Engineering and Applications , Qu\u00e9bec ,","place":["Canada"]},{"name":"CGCC, Center in Green Chemistry and Catalysis , Montr\u00e9al ,","place":["Canada"]},{"name":"Department of Biochemistry and Molecular Medicine, Universit\u00e9 de Montr\u00e9al , Montr\u00e9al ,","place":["Canada"]},{"name":"Department of Computer Science, University of Haifa , Haifa ,","place":["Israel"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stella","family":"Cellier-Goetghebeur","sequence":"additional","affiliation":[{"name":"PROTEO, The Qu\u00e9bec Network for Research on Protein, Function, Engineering and Applications , Qu\u00e9bec ,","place":["Canada"]},{"name":"CGCC, Center in Green Chemistry and Catalysis , Montr\u00e9al ,","place":["Canada"]},{"name":"Department of Biochemistry and Molecular Medicine, Universit\u00e9 de Montr\u00e9al , Montr\u00e9al ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maxime","family":"St-Aubin","sequence":"additional","affiliation":[{"name":"PROTEO, The Qu\u00e9bec Network for Research on Protein, Function, Engineering and Applications , Qu\u00e9bec ,","place":["Canada"]},{"name":"CGCC, Center in Green Chemistry and Catalysis , Montr\u00e9al ,","place":["Canada"]},{"name":"Department of Biochemistry and Molecular Medicine, Universit\u00e9 de Montr\u00e9al , Montr\u00e9al ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keigo","family":"Ide","sequence":"additional","affiliation":[{"name":"bitBiome , Tokyo ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janine N","family":"Copp","sequence":"additional","affiliation":[{"name":"Michael Smith Laboratories, University of British Columbia , Vancouver ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soichiro","family":"Tsuda","sequence":"additional","affiliation":[{"name":"bitBiome , Tokyo ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nir","family":"Ben-Tal","sequence":"additional","affiliation":[{"name":"School of Neurobiology, Biochemistry and Biophysics, George S. Wise Faculty of Life Sciences, Tel Aviv University , Tel Aviv ,","place":["Israel"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8523-1614","authenticated-orcid":false,"given":"Rachel","family":"Kolodny","sequence":"additional","affiliation":[{"name":"Department of Computer Science, University of Haifa , Haifa ,","place":["Israel"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joelle N","family":"Pelletier","sequence":"additional","affiliation":[{"name":"PROTEO, The Qu\u00e9bec Network for Research on Protein, Function, Engineering and Applications , Qu\u00e9bec ,","place":["Canada"]},{"name":"CGCC, Center in Green Chemistry and Catalysis , Montr\u00e9al ,","place":["Canada"]},{"name":"Department of Biochemistry and Molecular Medicine, Universit\u00e9 de Montr\u00e9al , Montr\u00e9al ,","place":["Canada"]},{"name":"Chemistry Department, Universit\u00e9 de Montr\u00e9al , Montr\u00e9al ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,9,16]]},"reference":[{"key":"2025101300113993700_msaf215-B1","doi-asserted-by":"publisher","first-page":"D517","DOI":"10.1093\/nar\/gkz935","article-title":"CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database","volume":"48","author":"Alcock","year":"2019","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B2","doi-asserted-by":"publisher","first-page":"396","DOI":"10.1021\/acs.biochem.2c00513","article-title":"Tuning selectivity in CalA lipase: beyond tunnel engineering","volume":"62","author":"Alejaldre","year":"2023","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B3","doi-asserted-by":"publisher","first-page":"3389","DOI":"10.1093\/nar\/25.17.3389","article-title":"Gapped BLAST and PSI-BLAST: a new generation of protein database search programs","volume":"25","author":"Altschul","year":"1997","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B4","doi-asserted-by":"publisher","first-page":"5134","DOI":"10.1093\/molbev\/msab240","article-title":"Fold evolution before LUCA: common ancestry of SH3 domains and OB domains","volume":"38","author":"Alvarez-Carre\u00f1o","year":"2021","journal-title":"Mol Biol Evol."},{"key":"2025101300113993700_msaf215-B5","doi-asserted-by":"publisher","first-page":"5267","DOI":"10.1021\/bi00695a006","article-title":"Purification and properties of Escherichia coli dihydrofolate reductase","volume":"14","author":"Baccanari","year":"1975","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B6","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1016\/S0176-1617(11)81515-4","article-title":"Biochemical evidence for the presence of a bifunctional dihydrofolate reductase-thymidylate synthase in plant species","volume":"147","author":"Balestrazzi","year":"1995","journal-title":"J Plant Physiol."},{"key":"2025101300113993700_msaf215-B7","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1016\/0092-8674(93)80076-Q","article-title":"Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12","volume":"75","author":"Blakely","year":"1993","journal-title":"Cell"},{"key":"2025101300113993700_msaf215-B8","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1038\/188231a0","article-title":"Crystalline dihydropteroylglutamic acid","volume":"188","author":"Blakley","year":"1960","journal-title":"Nature"},{"key":"2025101300113993700_msaf215-B9","doi-asserted-by":"publisher","first-page":"14988","DOI":"10.1021\/bi020453b","article-title":"Kinetic analysis of R67 dihydrofolate reductase folding: from the unfolded monomer to the native tetramer","volume":"41","author":"Bodenreider","year":"2002","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B10","doi-asserted-by":"publisher","first-page":"11414","DOI":"10.1021\/bi960205d","article-title":"Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein","volume":"35","author":"Bradrick","year":"1996","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B11","doi-asserted-by":"publisher","first-page":"1187","DOI":"10.1016\/j.jmb.2003.11.049","article-title":"Structural and biochemical analysis of sliding clamp\/ligand interactions suggest a competition between replicative and translesion DNA polymerases","volume":"335","author":"Burnouf","year":"2004","journal-title":"J Mol Biol."},{"key":"2025101300113993700_msaf215-B12","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1111\/j.1469-0691.2008.02679.x","article-title":"Antibiotic resistance genes from the environment: a perspective through newly identified antibiotic resistance mechanisms in the clinical setting","volume":"15","author":"Cante\u00f3n","year":"2009","journal-title":"Clin Microbiol Infect."},{"key":"2025101300113993700_msaf215-B13","doi-asserted-by":"publisher","first-page":"721","DOI":"10.1146\/annurev.bi.64.070195.003445","article-title":"The catalytic mechanism and structure of thymidylate synthase","volume":"64","author":"Carreras","year":"1995","journal-title":"Annu Rev Biochem."},{"key":"2025101300113993700_msaf215-B14","doi-asserted-by":"publisher","first-page":"1768","DOI":"10.3390\/antibiotics11121768","article-title":"Discovery of highly trimethoprim-resistant DfrB dihydrofolate reductases in diverse environmental settings suggests an evolutionary advantage unrelated to antibiotic resistance","volume":"11","author":"Cellier-Goetghebeur","year":"2022","journal-title":"Antibiotics"},{"key":"2025101300113993700_msaf215-B15","doi-asserted-by":"publisher","first-page":"e1003926","DOI":"10.1371\/journal.pcbi.1003926","article-title":"ECOD: an evolutionary classification of protein domains","volume":"10","author":"Cheng","year":"2014","journal-title":"PLoS Comput Biol."},{"key":"2025101300113993700_msaf215-B16","doi-asserted-by":"publisher","first-page":"6596","DOI":"10.1021\/bi052504l","article-title":"Effects of temperature and viscosity on R67 dihydrofolate reductase catalysis","volume":"45","author":"Chopra","year":"2006","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B17","doi-asserted-by":"publisher","first-page":"542","DOI":"10.1038\/s41589-018-0043-2","article-title":"Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein","volume":"14","author":"Clifton","year":"2018","journal-title":"Nat Chem Biol."},{"key":"2025101300113993700_msaf215-B18","doi-asserted-by":"publisher","first-page":"e52283","DOI":"10.1371\/journal.pone.0052283","article-title":"Chimeric \u03b2-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions","volume":"7","author":"Clouthier","year":"2012","journal-title":"PLoS One"},{"key":"2025101300113993700_msaf215-B19","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/j.tibs.2014.12.004","article-title":"An evolutionary biochemist's perspective on promiscuity","volume":"40","author":"Copley","year":"2015","journal-title":"Trends Biochem Sci."},{"key":"2025101300113993700_msaf215-B20","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1110\/ps.03309504","article-title":"Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations","volume":"13","author":"Dam","year":"2004","journal-title":"Protein Sci"},{"key":"2025101300113993700_msaf215-B21","doi-asserted-by":"publisher","first-page":"324","DOI":"10.1016\/S0968-0004(98)01274-2","article-title":"Conservation of gene order: a fingerprint of proteins that physically interact","volume":"23","author":"Dandekar","year":"1998","journal-title":"Trends Biochem Sci."},{"key":"2025101300113993700_msaf215-B22","doi-asserted-by":"publisher","first-page":"772","DOI":"10.1016\/j.tibs.2022.04.005","article-title":"SRC homology 3 domains: multifaceted binding modules","volume":"47","author":"Dionne","year":"2022","journal-title":"Trends Biochem Sci."},{"key":"2025101300113993700_msaf215-B23","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1021\/acs.biochem.5b00981","article-title":"Tales of dihydrofolate binding to R67 dihydrofolate reductase","volume":"55","author":"Duff","year":"2016","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B24","doi-asserted-by":"publisher","first-page":"e1002195","DOI":"10.1371\/journal.pcbi.1002195","article-title":"Accelerated profile HMM searches","volume":"7","author":"Eddy","year":"2011","journal-title":"PLoS Comput Biol."},{"key":"2025101300113993700_msaf215-B25","doi-asserted-by":"publisher","author":"Evans","year":"2021  10","DOI":"10.1101\/2021.10.04.463034"},{"key":"2025101300113993700_msaf215-B26","doi-asserted-by":"publisher","first-page":"5802","DOI":"10.1073\/pnas.77.10.5802","article-title":"Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata","volume":"77","author":"Ferone","year":"1980","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2025101300113993700_msaf215-B27","doi-asserted-by":"publisher","first-page":"726","DOI":"10.1136\/bmj.1.5802.726","article-title":"Trimethoprim resistance determined by R factors","volume":"1","author":"Fleming","year":"1972","journal-title":"BMJ"},{"key":"2025101300113993700_msaf215-B28","doi-asserted-by":"publisher","first-page":"3150","DOI":"10.1093\/bioinformatics\/bts565","article-title":"CD-HIT: accelerated for clustering the next-generation sequencing data","volume":"28","author":"Fu","year":"2012","journal-title":"Bioinformatics"},{"key":"2025101300113993700_msaf215-B29","doi-asserted-by":"publisher","first-page":"D274","DOI":"10.1093\/nar\/gkaa1018","article-title":"COG database update: focus on microbial diversity, model organisms, and widespread pathogens","volume":"49","author":"Galperin","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B30","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1038\/s41589-018-0064-x","article-title":"Enzymes emerge by upcycling","volume":"14","author":"Harms","year":"2018","journal-title":"Nat Chem Biol."},{"key":"2025101300113993700_msaf215-B31","doi-asserted-by":"publisher","first-page":"46995","DOI":"10.1074\/jbc.M404484200","article-title":"Role of lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations","volume":"279","author":"Hicks","year":"2004","journal-title":"J Biol Chem."},{"key":"2025101300113993700_msaf215-B32","doi-asserted-by":"publisher","first-page":"590","DOI":"10.1002\/cbic.200400237","article-title":"Searching sequence space: two different approaches to dihydrofolate reductase catalysis","volume":"6","author":"Howell","year":"2005","journal-title":"ChemBioChem"},{"key":"2025101300113993700_msaf215-B33","doi-asserted-by":"publisher","first-page":"1591","DOI":"10.1096\/fasebj.4.6.2180768","article-title":"Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa","volume":"4","author":"Ivanetich","year":"1990","journal-title":"FASEB J"},{"key":"2025101300113993700_msaf215-B34","doi-asserted-by":"publisher","first-page":"12420","DOI":"10.1021\/bi050881s","article-title":"Calorimetric studies of ligand binding in R67 dihydrofolate reductase","volume":"44","author":"Jackson","year":"2005","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B35","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"2025101300113993700_msaf215-B36","doi-asserted-by":"publisher","first-page":"112","DOI":"10.1002\/pro.3280","article-title":"Improvements to the APBS biomolecular solvation software suite","volume":"27","author":"Jurrus","year":"2018","journal-title":"Protein Sci"},{"key":"2025101300113993700_msaf215-B37","doi-asserted-by":"publisher","first-page":"548","DOI":"10.1038\/s41589-018-0042-3","article-title":"Evolution of chalcone isomerase from a noncatalytic ancestor","volume":"14","author":"Kaltenbach","year":"2018","journal-title":"Nat Chem Biol."},{"key":"2025101300113993700_msaf215-B38","doi-asserted-by":"publisher","first-page":"9078","DOI":"10.1021\/bi1007222","article-title":"The tail wagging the dog: insights into catalysis in R67 dihydrofolate reductase","volume":"49","author":"Kamath","year":"2010","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B39","doi-asserted-by":"publisher","first-page":"772","DOI":"10.1093\/molbev\/mst010","article-title":"MAFFT multiple sequence alignment software version 7: improvements in performance and usability","volume":"30","author":"Katoh","year":"2013","journal-title":"Mol Biol Evol."},{"key":"2025101300113993700_msaf215-B40","doi-asserted-by":"publisher","first-page":"16","DOI":"10.1016\/j.jsb.2014.09.001","article-title":"Repurposing TRASH: emergence of the enzyme organomercurial lyase from a non-catalytic zinc finger scaffold","volume":"188","author":"Kaur","year":"2014","journal-title":"J Struct Biol."},{"key":"2025101300113993700_msaf215-B41","doi-asserted-by":"publisher","first-page":"498","DOI":"10.1016\/j.cbpa.2006.08.011","article-title":"Enzyme promiscuity: evolutionary and mechanistic aspects","volume":"10","author":"Khersonsky","year":"2006","journal-title":"Curr Opin Chem Biol."},{"key":"2025101300113993700_msaf215-B42","doi-asserted-by":"publisher","first-page":"143","DOI":"10.2174\/1389203053545444","article-title":"SH3-like fold proteins are structurally conserved and functionally divergent","volume":"6","author":"Kishan","year":"2005","journal-title":"Curr Protein Pept Sci."},{"key":"2025101300113993700_msaf215-B43","doi-asserted-by":"publisher","first-page":"1455","DOI":"10.1038\/s41396-023-01460-7","article-title":"Trimethoprim resistance in surface and wastewater is mediated by contrasting variants of the dfrB gene","volume":"17","author":"Kneis","year":"2023","journal-title":"ISME J"},{"key":"2025101300113993700_msaf215-B44","doi-asserted-by":"publisher","first-page":"14913","DOI":"10.1021\/acs.est.2c00370","article-title":"Antibiotic resistance genes in river biofilms: a metagenomic approach toward the identification of sources and candidate hosts","volume":"56","author":"Kneis","year":"2022","journal-title":"Environ Sci Technol."},{"key":"2025101300113993700_msaf215-B45","doi-asserted-by":"publisher","first-page":"wrae243","DOI":"10.1093\/ismejo\/wrae243","article-title":"Bacteria of the order Burkholderiales are original environmental hosts of type II trimethoprim resistance genes (dfrB)","volume":"18","author":"Kneis","year":"2024","journal-title":"ISME J"},{"key":"2025101300113993700_msaf215-B46","doi-asserted-by":"publisher","first-page":"14878","DOI":"10.1021\/bi701532r","article-title":"Crystal structure of a type II dihydrofolate reductase catalytic ternary complex","volume":"46","author":"Krahn","year":"2007","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B47","doi-asserted-by":"publisher","first-page":"657","DOI":"10.1111\/j.1365-313X.1993.00657.x","article-title":"The origin of the bifunctional dihydrofolate reductasethymidylate synthase isogenes of Arabidopsis thaliana","volume":"3","author":"Lazar","year":"1993","journal-title":"Plant J"},{"key":"2025101300113993700_msaf215-B48","doi-asserted-by":"publisher","first-page":"20220040","DOI":"10.1098\/rstb.2022.0040","article-title":"A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype","volume":"378","author":"Lemay-St-Denis","year":"2023","journal-title":"Philos Trans R Soc B Biol Sci"},{"key":"2025101300113993700_msaf215-B49","doi-asserted-by":"publisher","first-page":"433","DOI":"10.3390\/antibiotics10040433","article-title":"The bacterial genomic context of highly trimethoprim-resistant DfrB dihydrofolate reductases highlights an emerging threat to public health","volume":"10","author":"Lemay-St-Denis","year":"2021","journal-title":"Antibiotics"},{"key":"2025101300113993700_msaf215-B50","doi-asserted-by":"publisher","first-page":"12560","DOI":"10.1039\/D3CC04209J","article-title":"From a binding module to essential catalytic activity: how nature stumbled on a good thing","volume":"59","author":"Lemay-St-Denis","year":"2023","journal-title":"Chem Commun"},{"key":"2025101300113993700_msaf215-B51","doi-asserted-by":"publisher","first-page":"W242","DOI":"10.1093\/nar\/gkw290","article-title":"Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees","volume":"44","author":"Letunic","year":"2016","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B52","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/978-1-4614-7780-8_1","volume-title":"Lateral gene transfer in evolution","author":"Levin","year":"2013"},{"key":"2025101300113993700_msaf215-B53","doi-asserted-by":"publisher","first-page":"1262","DOI":"10.1038\/nature06942","article-title":"Assembly reflects evolution of protein complexes","volume":"453","author":"Levy","year":"2008","journal-title":"Nature"},{"key":"2025101300113993700_msaf215-B54","doi-asserted-by":"publisher","first-page":"4242","DOI":"10.1021\/bi0026425","article-title":"Interligand overhauser effects in type II dihydrofolate reductase","volume":"40","author":"Li","year":"2001","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B55","doi-asserted-by":"publisher","first-page":"D170","DOI":"10.1093\/nar\/gkw1081","article-title":"Uniclust databases of clustered and deeply annotated protein sequences and alignments","volume":"45","author":"Mirdita","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B56","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold: making protein folding accessible to all","volume":"19","author":"Mirdita","year":"2022","journal-title":"Nat Methods."},{"key":"2025101300113993700_msaf215-B57","doi-asserted-by":"publisher","first-page":"D412","DOI":"10.1093\/nar\/gkaa913","article-title":"Pfam: the protein families database in 2021","volume":"49","author":"Mistry","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B58","doi-asserted-by":"publisher","first-page":"1018","DOI":"10.1038\/nsb1195-1018","article-title":"A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site","volume":"2","author":"Narayana","year":"1995","journal-title":"Nat Struct Mol Biol."},{"key":"2025101300113993700_msaf215-B59","doi-asserted-by":"publisher","first-page":"700","DOI":"10.3390\/microorganisms10040700","article-title":"IntegronFinder 2.0: identification and analysis of integrons across bacteria, with a focus on antibiotic resistance in Klebsiella","volume":"10","author":"N\u00e9ron","year":"2022","journal-title":"Microorganisms"},{"key":"2025101300113993700_msaf215-B60","doi-asserted-by":"publisher","first-page":"268","DOI":"10.1093\/molbev\/msu300","article-title":"IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies","volume":"32","author":"Nguyen","year":"2015","journal-title":"Mol Biol Evol."},{"key":"2025101300113993700_msaf215-B61","doi-asserted-by":"publisher","first-page":"1695","DOI":"10.1021\/bi00058a002","article-title":"Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer .tautm. two-dimer equilibrium","volume":"32","author":"Nichols","year":"1993","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B62","doi-asserted-by":"publisher","first-page":"92","DOI":"10.1038\/s43705-022-00179-4","article-title":"Validation of the application of gel beads-based single-cell genome sequencing platform to soil and seawater","volume":"2","author":"Nishikawa","year":"2022","journal-title":"ISME Commun"},{"key":"2025101300113993700_msaf215-B63","doi-asserted-by":"publisher","first-page":"1101","DOI":"10.1136\/bmj.2.5312.1101","article-title":"Successful treatment of a case of Proteus septicaemia","volume":"2","author":"Noall","year":"1962","journal-title":"BMJ"},{"key":"2025101300113993700_msaf215-B64","doi-asserted-by":"publisher","first-page":"710","DOI":"10.1038\/s41586-024-07809-y","article-title":"Birth of protein folds and functions in the virome","volume":"633","author":"Nomburg","year":"2024","journal-title":"Nature"},{"key":"2025101300113993700_msaf215-B65","doi-asserted-by":"publisher","first-page":"R91","DOI":"10.1016\/S1074-5521(99)80033-7","article-title":"Catalytic promiscuity and the evolution of new enzymatic activities","volume":"6","author":"O\u2019Brien","year":"1999","journal-title":"Chem Biol."},{"key":"2025101300113993700_msaf215-B66","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1038\/nature20159","article-title":"An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme","volume":"539","author":"Ortmayer","year":"2016","journal-title":"Nature"},{"key":"2025101300113993700_msaf215-B67","doi-asserted-by":"publisher","first-page":"1415","DOI":"10.1093\/protein\/10.12.1415","article-title":"A glutamine 67\u2013&gt; histidine mutation in homotetrameric R67 dihydrofolate reductase results in four mutations per single active site pore and causes substantial substrate and cofactor inhibition","volume":"10","author":"Park","year":"1997","journal-title":"Protein Eng Des Sel."},{"key":"2025101300113993700_msaf215-B68","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1007\/s00203-023-03424-z","article-title":"Global profiling of antibiotic resistomes in maize rhizospheres","volume":"205","author":"Pham","year":"2023","journal-title":"Arch Microbiol."},{"key":"2025101300113993700_msaf215-B69","doi-asserted-by":"publisher","first-page":"10895","DOI":"10.1021\/bi00109a013","article-title":"Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein","volume":"30","author":"Reece","year":"1991","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B70","doi-asserted-by":"publisher","first-page":"D753","DOI":"10.1093\/nar\/gkac1080","article-title":"MGnify: the microbiome sequence data analysis resource in 2023","volume":"51","author":"Richardson","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B71","doi-asserted-by":"publisher","first-page":"809","DOI":"10.1093\/protein\/gzh090","article-title":"Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations","volume":"17","author":"Schmitzer","year":"2004","journal-title":"Protein Eng Des Sel."},{"key":"2025101300113993700_msaf215-B72","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1146\/annurev.biophys.33.110502.133613","article-title":"Structure, dynamics, and catalytic function of dihydrofolate reductase","volume":"33","author":"Schnell","year":"2004","journal-title":"Annu Rev Biophys Biomol Struct."},{"key":"2025101300113993700_msaf215-B73","doi-asserted-by":"publisher","first-page":"2068","DOI":"10.1093\/bioinformatics\/btu153","article-title":"Prokka: rapid prokaryotic genome annotation","volume":"30","author":"Seemann","year":"2014","journal-title":"Bioinformatics"},{"key":"2025101300113993700_msaf215-B74","doi-asserted-by":"publisher","first-page":"2498","DOI":"10.1101\/gr.1239303","article-title":"Cytoscape: a software environment for integrated models of biomolecular interaction networks","volume":"13","author":"Shannon","year":"2003","journal-title":"Genome Res"},{"key":"2025101300113993700_msaf215-B75","doi-asserted-by":"publisher","first-page":"15664","DOI":"10.1021\/bi026676j","article-title":"Breaking symmetry: mutations engineered into R67 dihydrofolate reductase, a D2 symmetric homotetramer possessing a single active site pore","volume":"41","author":"Smiley","year":"2002","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B76","doi-asserted-by":"publisher","first-page":"4212","DOI":"10.1021\/bi961794q","article-title":"Kinetic scheme for thymidylate synthase from Escherichia coli: determination from measurements of ligand binding, primary and secondary isotope effects, and pre-steady-state catalysis","volume":"36","author":"Spencer","year":"1997","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B77","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1126\/science.1071196","article-title":"Rooting the eukaryote tree by using a derived gene fusion","volume":"297","author":"Stechmann","year":"2002","journal-title":"Science"},{"key":"2025101300113993700_msaf215-B78","doi-asserted-by":"publisher","first-page":"473","DOI":"10.1186\/s12859-019-3019-7","article-title":"HH-suite3 for fast remote homology detection and deep protein annotation","volume":"20","author":"Steinegger","year":"2019","journal-title":"BMC Bioinform"},{"key":"2025101300113993700_msaf215-B79","doi-asserted-by":"publisher","first-page":"1026","DOI":"10.1038\/nbt.3988","article-title":"MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets","volume":"35","author":"Steinegger","year":"2017","journal-title":"Nat Biotechnol."},{"key":"2025101300113993700_msaf215-B80","doi-asserted-by":"publisher","first-page":"926","DOI":"10.1093\/bioinformatics\/btu739","article-title":"UniRef clusters: a comprehensive and scalable alternative for improving sequence similarity searches","volume":"31","author":"Suzek","year":"2015","journal-title":"Bioinformatics"},{"key":"2025101300113993700_msaf215-B81","doi-asserted-by":"publisher","first-page":"1533","DOI":"10.1093\/molbev\/msz086","article-title":"High-order epistasis in catalytic power of dihydrofolate reductase gives rise to a rugged fitness landscape in the presence of trimethoprim selection","volume":"36","author":"Tamer","year":"2019","journal-title":"Mol Biol Evol"},{"key":"2025101300113993700_msaf215-B82","doi-asserted-by":"publisher","first-page":"W345","DOI":"10.1093\/nar\/gkac247","article-title":"The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2022 update","volume":"50","author":"The Galaxy Community","year":"2022","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B83","doi-asserted-by":"publisher","first-page":"D523","DOI":"10.1093\/nar\/gkac1052","article-title":"UniProt: the universal protein knowledgebase in 2023","volume":"51","author":"The UniProt Consortium","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2025101300113993700_msaf215-B84","doi-asserted-by":"publisher","first-page":"2261","DOI":"10.1021\/acsmedchemlett.0c00393","article-title":"Dual-target inhibitors of the folate pathway inhibit intrinsically trimethoprim-resistant DfrB dihydrofolate reductases","volume":"11","author":"Toulouse","year":"2020","journal-title":"ACS Med Chem Lett."},{"key":"2025101300113993700_msaf215-B85","doi-asserted-by":"publisher","first-page":"6366","DOI":"10.1021\/bi952923q","article-title":"Heterologous expression and characterization of the bifunctional dihydrofolate reductase\u2212thymidylate synthase enzyme of Toxoplasma gondii","volume":"35","author":"Trujillo","year":"1996","journal-title":"Biochemistry"},{"key":"2025101300113993700_msaf215-B86","doi-asserted-by":"publisher","first-page":"243","DOI":"10.1038\/s41587-023-01773-0","article-title":"Fast and accurate protein structure search with foldseek","volume":"42","author":"Van Kempen","year":"2024","journal-title":"Nat Biotechnol."},{"key":"2025101300113993700_msaf215-B87","doi-asserted-by":"publisher","author":"Wallner","DOI":"10.1093\/bioinformatics\/btad573"},{"key":"2025101300113993700_msaf215-B88","doi-asserted-by":"crossref","DOI":"10.1007\/978-3-319-24277-4","volume-title":"Ggplot2: elegant graphics for data analysis","author":"Wickham","year":"2016","edition":"2nd ed"},{"key":"2025101300113993700_msaf215-B89","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1038\/nprot.2007.521","article-title":"Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances","volume":"3","author":"Wiegand","year":"2008","journal-title":"Nat Protoc."},{"key":"2025101300113993700_msaf215-B90","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1038\/nsb921","article-title":"Insights into antifolate resistance from malarial DHFR-TS structures","volume":"10","author":"Yuvaniyama","year":"2003","journal-title":"Nat Struct Mol Biol."}],"container-title":["Molecular Biology and Evolution"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/mbe\/advance-article-pdf\/doi\/10.1093\/molbev\/msaf215\/64288525\/msaf215.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/mbe\/article-pdf\/42\/10\/msaf215\/64288525\/msaf215.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/mbe\/article-pdf\/42\/10\/msaf215\/64288525\/msaf215.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T04:11:50Z","timestamp":1760328710000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/mbe\/article\/doi\/10.1093\/molbev\/msaf215\/8256353"}},"subtitle":[],"editor":[{"given":"Klara","family":"Hlouchova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,9,16]]},"references-count":90,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2025,10,1]]}},"URL":"https:\/\/doi.org\/10.1093\/molbev\/msaf215","relation":{},"ISSN":["0737-4038","1537-1719"],"issn-type":[{"value":"0737-4038","type":"print"},{"value":"1537-1719","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2025,10]]},"published":{"date-parts":[[2025,9,16]]},"article-number":"msaf215"}}