ID DYR21_ECOLX Reviewed; 78 AA. AC P00383; DT 21-JUL-1986, integrated into UniProtKB/Swiss-Prot. DT 21-JUL-1986, sequence version 1. DT 28-JUN-2023, entry version 110. DE RecName: Full=Dihydrofolate reductase type 2; DE EC=1.5.1.3; DE AltName: Full=Dihydrofolate reductase type II; OS Escherichia coli. OG Plasmid R67. OC Bacteria; Pseudomonadota; Gammaproteobacteria; Enterobacterales; OC Enterobacteriaceae; Escherichia. OX NCBI_TaxID=562; RN [1] RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RX PubMed=6735180; DOI=10.1016/0378-1119(84)90266-x; RA Brisson N., Hohn T.; RT "Nucleotide sequence of the dihydrofolate-reductase gene borne by the RT plasmid R67 and conferring methotrexate resistance."; RL Gene 28:271-275(1984). RN [2] RP PROTEIN SEQUENCE. RX PubMed=387758; DOI=10.1016/s0021-9258(19)86600-0; RA Stone D., Smith S.L.; RT "The amino acid sequence of the trimethoprim-resistant dihydrofolate RT reductase specified in Escherichia coli by R-plasmid R67."; RL J. Biol. Chem. 254:10857-10861(1979). RN [3] RP BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, IDENTIFICATION BY MASS RP SPECTROMETRY, AND MUTAGENESIS OF SER-65; GLN-67; ILE-68 AND TYR-69. RX PubMed=11560482; DOI=10.1021/bi0110544; RA Strader M.B., Smiley R.D., Stinnett L.G., VerBerkmoes N.C., Howell E.E.; RT "Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 RT dihydrofolate reductase."; RL Biochemistry 40:11344-11352(2001). RN [4] RP X-RAY CRYSTALLOGRAPHY (1.7 ANGSTROMS) OF 17-78 IN COMPLEX WITH FOLATE, RP DOMAIN, AND SUBUNIT. RX PubMed=7583655; DOI=10.1038/nsb1195-1018; RA Narayana N., Matthews D.A., Howell E.E., Nguyen-Huu X.; RT "A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant RT bacteria has a novel D2-symmetric active site."; RL Nat. Struct. Biol. 2:1018-1025(1995). RN [5] RP X-RAY CRYSTALLOGRAPHY (1.1 ANGSTROMS) OF 17-78 OF APOPROTEIN, DOMAIN, AND RP SUBUNIT. RX PubMed=16790925; DOI=10.1107/s0907444906014764; RA Narayana N.; RT "High-resolution structure of a plasmid-encoded dihydrofolate reductase: RT pentagonal network of water molecules in the D2-symmetric active site."; RL Acta Crystallogr. D 62:695-706(2006). RN [6] RP X-RAY CRYSTALLOGRAPHY (0.96 ANGSTROMS) OF 17-78 IN COMPLEX WITH RP DIHYDROFOLATE AND NADP. RX PubMed=18052202; DOI=10.1021/bi701532r; RA Krahn J.M., Jackson M.R., DeRose E.F., Howell E.E., London R.E.; RT "Crystal structure of a type II dihydrofolate reductase catalytic ternary RT complex."; RL Biochemistry 46:14878-14888(2007). RN [7] RP X-RAY CRYSTALLOGRAPHY (1.15 ANGSTROMS) OF 17-78 OF MUTANT HIS-67 IN COMPLEX RP WITH NADP. RX PubMed=17473013; DOI=10.1110/ps.062740907; RA Divya N., Grifith E., Narayana N.; RT "Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex RT reveals a novel cofactor binding mode."; RL Protein Sci. 16:1063-1068(2007). CC -!- FUNCTION: Key enzyme in folate metabolism. Catalyzes an essential CC reaction for de novo glycine and purine synthesis, and for DNA CC precursor synthesis. CC -!- CATALYTIC ACTIVITY: CC Reaction=(6S)-5,6,7,8-tetrahydrofolate + NADP(+) = 7,8-dihydrofolate + CC H(+) + NADPH; Xref=Rhea:RHEA:15009, ChEBI:CHEBI:15378, CC ChEBI:CHEBI:57451, ChEBI:CHEBI:57453, ChEBI:CHEBI:57783, CC ChEBI:CHEBI:58349; EC=1.5.1.3; CC -!- BIOPHYSICOCHEMICAL PROPERTIES: CC Kinetic parameters: CC KM=5.8 uM for dihydrofolate {ECO:0000269|PubMed:11560482}; CC KM=3 uM for NADPH {ECO:0000269|PubMed:11560482}; CC -!- PATHWAY: Cofactor biosynthesis; tetrahydrofolate biosynthesis; 5,6,7,8- CC tetrahydrofolate from 7,8-dihydrofolate: step 1/1. CC -!- SUBUNIT: Homotetramer. {ECO:0000269|PubMed:11560482, CC ECO:0000269|PubMed:16790925, ECO:0000269|PubMed:17473013, CC ECO:0000269|PubMed:18052202, ECO:0000269|PubMed:7583655}. CC -!- DOMAIN: The active site is situated at the inner surface of a pore CC formed by the four subunits. {ECO:0000269|PubMed:16790925, CC ECO:0000269|PubMed:7583655}. CC -!- MISCELLANEOUS: Type II plasmid-specified enzyme is practically CC insensitive to trimethoprim and methotrexate. CC --------------------------------------------------------------------------- CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms CC Distributed under the Creative Commons Attribution (CC BY 4.0) License CC --------------------------------------------------------------------------- DR EMBL; K02118; AAA26083.1; -; Genomic_DNA. DR PIR; A91512; RDECD6. DR RefSeq; WP_000442373.1; NZ_OW968113.1. DR PDB; 1VIE; X-ray; 1.70 A; A=17-78. DR PDB; 1VIF; X-ray; 1.80 A; A=17-78. DR PDB; 2GQV; X-ray; 1.10 A; A=17-78. DR PDB; 2P4T; X-ray; 1.15 A; A=17-78. DR PDB; 2RH2; X-ray; 0.96 A; A=17-78. DR PDB; 2RK1; X-ray; 1.26 A; A=17-78. DR PDB; 2RK2; X-ray; 1.90 A; A=17-78. DR PDB; 3SFM; X-ray; 1.40 A; A=17-78. DR PDB; 6NXZ; X-ray; 1.75 A; A=17-78. DR PDB; 6NY0; X-ray; 1.40 A; A=18-78. DR PDBsum; 1VIE; -. DR PDBsum; 1VIF; -. DR PDBsum; 2GQV; -. DR PDBsum; 2P4T; -. DR PDBsum; 2RH2; -. DR PDBsum; 2RK1; -. DR PDBsum; 2RK2; -. DR PDBsum; 3SFM; -. DR PDBsum; 6NXZ; -. DR PDBsum; 6NY0; -. DR AlphaFoldDB; P00383; -. DR BMRB; P00383; -. DR SMR; P00383; -. DR BRENDA; 1.5.1.3; 2026. DR SABIO-RK; P00383; -. DR UniPathway; UPA00077; UER00158. DR EvolutionaryTrace; P00383; -. DR GO; GO:0004146; F:dihydrofolate reductase activity; IEA:UniProtKB-EC. DR GO; GO:0006730; P:one-carbon metabolic process; IEA:UniProtKB-KW. DR GO; GO:0046677; P:response to antibiotic; IEA:UniProtKB-KW. DR GO; GO:0031427; P:response to methotrexate; IEA:UniProtKB-KW. DR GO; GO:0009410; P:response to xenobiotic stimulus; IEA:InterPro. DR GO; GO:0046654; P:tetrahydrofolate biosynthetic process; IEA:UniProtKB-UniPathway. DR Gene3D; 2.30.30.60; -; 1. DR InterPro; IPR009159; Dhfr_type_II. DR InterPro; IPR008990; Elect_transpt_acc-like_dom_sf. DR InterPro; IPR023408; MscS_beta-dom_sf. DR Pfam; PF06442; DHFR_2; 1. DR PIRSF; PIRSF000199; Dhfr_type_II; 1. DR SUPFAM; SSF50090; Electron transport accessory proteins; 1. PE 1: Evidence at protein level; KW 3D-structure; Antibiotic resistance; Direct protein sequencing; KW Methotrexate resistance; NADP; One-carbon metabolism; Oxidoreductase; KW Plasmid; Trimethoprim resistance. FT CHAIN 1..78 FT /note="Dihydrofolate reductase type 2" FT /id="PRO_0000186435" FT BINDING 32..36 FT /ligand="NADP(+)" FT /ligand_id="ChEBI:CHEBI:58349" FT /evidence="ECO:0000269|PubMed:17473013, FT ECO:0000269|PubMed:18052202, ECO:0007744|PDB:2P4T, FT ECO:0007744|PDB:2RK1, ECO:0007744|PDB:2RK2" FT BINDING 66..69 FT /ligand="NADP(+)" FT /ligand_id="ChEBI:CHEBI:58349" FT /evidence="ECO:0000269|PubMed:17473013, FT ECO:0000269|PubMed:18052202, ECO:0007744|PDB:2P4T, FT ECO:0007744|PDB:2RK1, ECO:0007744|PDB:2RK2" FT BINDING 68 FT /ligand="substrate" FT /evidence="ECO:0000269|PubMed:18052202, FT ECO:0007744|PDB:2RK1" FT MUTAGEN 65 FT /note="S->A: No effect." FT /evidence="ECO:0000269|PubMed:11560482" FT MUTAGEN 67 FT /note="Q->C: Decreases affinity for NADPH and dihydrofolate FT about 9-fold." FT /evidence="ECO:0000269|PubMed:11560482" FT MUTAGEN 67 FT /note="Q->H: Increases affinity for dihydrofolate 36-fold. FT Increases affinity for NADPH 110-fold." FT /evidence="ECO:0000269|PubMed:11560482" FT MUTAGEN 68 FT /note="I->L,M: Decreases affinity for dihydrofolate about FT 5-fold. Decreases affinity for NADPH about 7-fold." FT /evidence="ECO:0000269|PubMed:11560482" FT MUTAGEN 69 FT /note="Y->F: Decreases affinity for dihydrofolate about FT 9-fold. Decreases affinity for NADPH about 22-fold." FT /evidence="ECO:0000269|PubMed:11560482" FT MUTAGEN 69 FT /note="Y->H: Decreases affinity for dihydrofolate about FT 9-fold. Decreases affinity for NADPH about 60-fold." FT /evidence="ECO:0000269|PubMed:11560482" FT STRAND 29..36 FT /evidence="ECO:0007829|PDB:2RH2" FT STRAND 39..46 FT /evidence="ECO:0007829|PDB:2RH2" FT STRAND 52..62 FT /evidence="ECO:0007829|PDB:2RH2" FT STRAND 66..70 FT /evidence="ECO:0007829|PDB:2RH2" FT HELIX 71..73 FT /evidence="ECO:0007829|PDB:2RH2" FT STRAND 74..76 FT /evidence="ECO:0007829|PDB:2RH2" SQ SEQUENCE 78 AA; 8446 MW; 0BDB0B9146529417 CRC64; MERSSNEVSN PVAGNFVFPS NATFGMGDRV RKKSGAAWQG QIVGWYCTNL TPEGYAVESE AHPGSVQIYP VAALERIN //