T2E5_ECOLX
ID T2E5_ECOLX Reviewed; 245 AA.
AC P04390;
DT 20-MAR-1987, integrated into UniProtKB/Swiss-Prot.
DT 23-JAN-2007, sequence version 3.
DT 03-AUG-2022, entry version 149.
DE RecName: Full=Type II restriction enzyme EcoRV {ECO:0000303|PubMed:12654995};
DE Short=R.EcoRV;
DE EC=3.1.21.4 {ECO:0000269|PubMed:1647200};
DE AltName: Full=Endonuclease EcoRV;
DE AltName: Full=Type-2 restriction enzyme EcoRV;
GN Name=ecoRVR;
OS Escherichia coli.
OG Plasmid pLB1 {ECO:0000303|PubMed:6328432}, and
OG Plasmid pLG13 {ECO:0000303|Ref.2}.
OC Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacterales;
OC Enterobacteriaceae; Escherichia.
OX NCBI_TaxID=562;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 2-35; 38-40 AND
RP 235-245, AND FUNCTION.
RC STRAIN=J62; PLASMID=pLB1 {ECO:0000303|PubMed:6328432};
RX PubMed=6328432; DOI=10.1093/nar/12.8.3659;
RA Bougueleret L., Schwarzstein M., Tsugita A., Zabeau M.;
RT "Characterization of the genes coding for the Eco RV restriction and
RT modification system of Escherichia coli.";
RL Nucleic Acids Res. 12:3659-3676(1984).
RN [2]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION, AND CATALYTIC ACTIVITY.
RC PLASMID=pLG13 {ECO:0000303|Ref.2};
RA Kraev A.S., Kravets A.N., Chernov B.K., Skryabin K.G., Baev A.A.;
RT "The EcoRV restriction-modification system: genes, enzymes, synthetic
RT substrates.";
RL Mol. Biol. (Mosk.) 19:236-242(1985).
RN [3]
RP FUNCTION, CATALYTIC ACTIVITY, SUBUNIT, AND MUTAGENESIS OF ASN-70; PRO-73;
RP ASP-74; ASP-90; LYS-92; 183-SER--ASN-188; SER-183; ASN-185; THR-186;
RP THR-187; ASN-188; GLY-190 AND 216-TRP--LYS-245.
RX PubMed=1647200; DOI=10.1021/bi00240a011;
RA Thielking V., Selent U., Koehler E., Wolfes H., Pieper U., Geiger R.,
RA Urbanke C., Winkler F.K., Pingoud A.;
RT "Site-directed mutagenesis studies with EcoRV restriction endonuclease to
RT identify regions involved in recognition and catalysis.";
RL Biochemistry 30:6416-6422(1991).
RN [4]
RP NOMENCLATURE, AND SUBTYPE.
RX PubMed=12654995; DOI=10.1093/nar/gkg274;
RA Roberts R.J., Belfort M., Bestor T., Bhagwat A.S., Bickle T.A.,
RA Bitinaite J., Blumenthal R.M., Degtyarev S.K., Dryden D.T., Dybvig K.,
RA Firman K., Gromova E.S., Gumport R.I., Halford S.E., Hattman S.,
RA Heitman J., Hornby D.P., Janulaitis A., Jeltsch A., Josephsen J., Kiss A.,
RA Klaenhammer T.R., Kobayashi I., Kong H., Krueger D.H., Lacks S.,
RA Marinus M.G., Miyahara M., Morgan R.D., Murray N.E., Nagaraja V.,
RA Piekarowicz A., Pingoud A., Raleigh E., Rao D.N., Reich N., Repin V.E.,
RA Selker E.U., Shaw P.C., Stein D.C., Stoddard B.L., Szybalski W.,
RA Trautner T.A., Van Etten J.L., Vitor J.M., Wilson G.G., Xu S.Y.;
RT "A nomenclature for restriction enzymes, DNA methyltransferases, homing
RT endonucleases and their genes.";
RL Nucleic Acids Res. 31:1805-1812(2003).
RN [5] {ECO:0007744|PDB:1RVE, ECO:0007744|PDB:2RVE, ECO:0007744|PDB:4RVE}
RP X-RAY CRYSTALLOGRAPHY (3.0 ANGSTROMS) IN COMPLEX WITH DNA, SUBUNIT, AND
RP DNA-BINDING.
RX PubMed=8491171; DOI=10.2210/pdb4rve/pdb;
RA Winkler F.K., Banner D.W., Oefner C., Tsernoglou D., Brown R.,
RA Heathman S.P., Bryan R.K., Martin P.D., Petratos K., Wilson K.S.;
RT "The crystal structure of EcoRV endonuclease and of its complexes with
RT cognate and non-cognate DNA fragments.";
RL EMBO J. 12:1781-1795(1993).
RN [6]
RP X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
RX PubMed=7819264; DOI=10.1021/bi00002a036;
RA Kostrewa D., Winkler F.K.;
RT "Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic
RT study of complexes with substrate and product DNA at 2 A resolution.";
RL Biochemistry 34:683-696(1995).
RN [7]
RP X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
RX PubMed=9367757; DOI=10.1006/jmbi.1997.1315;
RA Perona J.J., Martin A.M.;
RT "Conformational transitions and structural deformability of EcoRV
RT endonuclease revealed by crystallographic analysis.";
RL J. Mol. Biol. 273:207-225(1997).
RN [8]
RP X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
RX PubMed=9705308; DOI=10.1074/jbc.273.34.21721;
RA Horton N.C., Perona J.J.;
RT "Recognition of flanking DNA sequences by EcoRV endonuclease involves
RT alternative patterns of water-mediated contacts.";
RL J. Biol. Chem. 273:21721-21729(1998).
RN [9]
RP X-RAY CRYSTALLOGRAPHY (2.1 ANGSTROMS).
RX PubMed=9545372; DOI=10.1006/jmbi.1998.1655;
RA Horton N.C., Perona J.J.;
RT "Role of protein-induced bending in the specificity of DNA recognition:
RT crystal structure of EcoRV endonuclease complexed with d(AAAGAT) +
RT d(ATCTT).";
RL J. Mol. Biol. 277:779-787(1998).
RN [10]
RP X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS) OF GLN-69 MUTANTS.
RX PubMed=10446231; DOI=10.1093/nar/27.17.3438;
RA Thomas M.P., Brady R.L., Halford S.E., Sessions R.B., Baldwin G.S.;
RT "Structural analysis of a mutational hot-spot in the EcoRV restriction
RT endonuclease: a catalytic role for a main chain carbonyl group.";
RL Nucleic Acids Res. 27:3438-3445(1999).
CC -!- FUNCTION: A P subtype restriction enzyme that recognizes the double-
CC stranded sequence 5'-GATATC-3' and cleaves after T-3.
CC {ECO:0000269|PubMed:1647200, ECO:0000269|PubMed:6328432,
CC ECO:0000269|Ref.2, ECO:0000303|PubMed:12654995}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=Endonucleolytic cleavage of DNA to give specific double-
CC stranded fragments with terminal 5'-phosphates.; EC=3.1.21.4;
CC Evidence={ECO:0000269|PubMed:1647200, ECO:0000269|Ref.2};
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Note=Binds 2 magnesium ions per subunit.;
CC -!- SUBUNIT: Homodimer. {ECO:0000269|PubMed:8491171,
CC ECO:0000305|PubMed:1647200}.
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DR EMBL; X00530; CAA25208.1; -; Genomic_DNA.
DR EMBL; M19941; AAA24615.1; -; Genomic_DNA.
DR PIR; A00784; NDECR5.
DR RefSeq; NP_863580.1; NC_005019.1.
DR RefSeq; WP_011117659.1; NZ_VUDS01000061.1.
DR RefSeq; YP_007316617.1; NC_019982.1.
DR PDB; 1AZ0; X-ray; 2.00 A; A/B=2-245.
DR PDB; 1AZ3; X-ray; 2.40 A; A/B=2-245.
DR PDB; 1AZ4; X-ray; 2.40 A; A/B=2-245.
DR PDB; 1B94; X-ray; 1.90 A; A/B=2-245.
DR PDB; 1B95; X-ray; 2.05 A; A/B=2-245.
DR PDB; 1B96; X-ray; 2.30 A; A/B=2-245.
DR PDB; 1B97; X-ray; 1.90 A; A/B=2-245.
DR PDB; 1BGB; X-ray; 2.00 A; A/B=2-245.
DR PDB; 1BSS; X-ray; 2.15 A; A/B=2-245.
DR PDB; 1BSU; X-ray; 2.00 A; A/B=2-245.
DR PDB; 1BUA; X-ray; 2.15 A; A/B=2-245.
DR PDB; 1EO3; X-ray; 2.00 A; A/B=1-245.
DR PDB; 1EO4; X-ray; 1.90 A; A/B=1-245.
DR PDB; 1EON; X-ray; 1.60 A; A/B=1-245.
DR PDB; 1EOO; X-ray; 2.16 A; A/B=1-245.
DR PDB; 1EOP; X-ray; 2.60 A; A/B=1-245.
DR PDB; 1RV5; X-ray; 2.10 A; A/B=2-245.
DR PDB; 1RVA; X-ray; 2.00 A; A/B=2-245.
DR PDB; 1RVB; X-ray; 2.10 A; A/B=2-245.
DR PDB; 1RVC; X-ray; 2.10 A; A/B=2-245.
DR PDB; 1RVE; X-ray; 2.50 A; A/B=1-245.
DR PDB; 1STX; X-ray; 2.10 A; A/B=2-245.
DR PDB; 1SUZ; X-ray; 1.80 A; A/B=2-245.
DR PDB; 1SX5; X-ray; 1.50 A; A/B=2-245.
DR PDB; 1SX8; X-ray; 2.15 A; A/B=2-245.
DR PDB; 2B0D; X-ray; 2.00 A; A/B=1-245.
DR PDB; 2B0E; X-ray; 1.90 A; A/B=1-245.
DR PDB; 2GE5; X-ray; 2.40 A; A/B=2-220.
DR PDB; 2RVE; X-ray; 3.00 A; A/B=2-245.
DR PDB; 4RVE; X-ray; 3.00 A; A/B/C=2-245.
DR PDB; 5F8A; X-ray; 1.76 A; A/B=2-245.
DR PDB; 5HLK; X-ray; 2.00 A; A/B=2-245.
DR PDBsum; 1AZ0; -.
DR PDBsum; 1AZ3; -.
DR PDBsum; 1AZ4; -.
DR PDBsum; 1B94; -.
DR PDBsum; 1B95; -.
DR PDBsum; 1B96; -.
DR PDBsum; 1B97; -.
DR PDBsum; 1BGB; -.
DR PDBsum; 1BSS; -.
DR PDBsum; 1BSU; -.
DR PDBsum; 1BUA; -.
DR PDBsum; 1EO3; -.
DR PDBsum; 1EO4; -.
DR PDBsum; 1EON; -.
DR PDBsum; 1EOO; -.
DR PDBsum; 1EOP; -.
DR PDBsum; 1RV5; -.
DR PDBsum; 1RVA; -.
DR PDBsum; 1RVB; -.
DR PDBsum; 1RVC; -.
DR PDBsum; 1RVE; -.
DR PDBsum; 1STX; -.
DR PDBsum; 1SUZ; -.
DR PDBsum; 1SX5; -.
DR PDBsum; 1SX8; -.
DR PDBsum; 2B0D; -.
DR PDBsum; 2B0E; -.
DR PDBsum; 2GE5; -.
DR PDBsum; 2RVE; -.
DR PDBsum; 4RVE; -.
DR PDBsum; 5F8A; -.
DR PDBsum; 5HLK; -.
DR AlphaFoldDB; P04390; -.
DR SMR; P04390; -.
DR DrugBank; DB08651; 3'-THIO-THYMIDINE-5'-PHOSPHATE.
DR EvolutionaryTrace; P04390; -.
DR PRO; PR:P04390; -.
DR GO; GO:0003677; F:DNA binding; IEA:UniProtKB-KW.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0009036; F:type II site-specific deoxyribonuclease activity; IEA:UniProtKB-EC.
DR GO; GO:0009307; P:DNA restriction-modification system; IEA:UniProtKB-KW.
DR Gene3D; 3.40.600.10; -; 1.
DR InterPro; IPR037057; DNA_rep_MutH/T2_RE_sf.
DR InterPro; IPR011335; Restrct_endonuc-II-like.
DR InterPro; IPR015314; Restrct_endonuc_II_EcoRV.
DR InterPro; IPR019755; Restrct_endonuc_II_EcoRV_Pbac.
DR Pfam; PF09233; Endonuc-EcoRV; 1.
DR PIRSF; PIRSF000995; RE_EcoRV; 1.
DR SUPFAM; SSF52980; SSF52980; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Direct protein sequencing; DNA-binding; Endonuclease;
KW Hydrolase; Magnesium; Metal-binding; Nuclease; Plasmid; Restriction system.
FT INIT_MET 1
FT /note="Removed"
FT /evidence="ECO:0000269|PubMed:6328432"
FT CHAIN 2..245
FT /note="Type II restriction enzyme EcoRV"
FT /id="PRO_0000077305"
FT ACT_SITE 74
FT ACT_SITE 90
FT ACT_SITE 92
FT BINDING 45
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT BINDING 74
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT BINDING 74
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="2"
FT BINDING 90
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /ligand_label="1"
FT MUTAGEN 70
FT /note="N->Q: Decrease in activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 73
FT /note="P->A,G: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 74
FT /note="D->A: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 74
FT /note="D->E: Decrease in activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 90
FT /note="D->A,N,E,T: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 92
FT /note="K->E: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 183..188
FT /note="Missing: Weak, non-specific phosphodiesterase
FT activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 183
FT /note="S->A,T: Decrease in activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 183
FT /note="S->I: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 185
FT /note="N->D,A,Q: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 186
FT /note="T->S,N: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 187
FT /note="T->S,N: No loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 188
FT /note="N->A,Q,T: Decrease in activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 188
FT /note="N->D: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 190
FT /note="G->A: No loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT MUTAGEN 216..245
FT /note="Missing: Loss of activity."
FT /evidence="ECO:0000269|PubMed:1647200"
FT HELIX 3..17
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 20..25
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 30..32
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 37..58
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 62..64
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 67..70
FT /evidence="ECO:0007829|PDB:1EO4"
FT STRAND 74..78
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 86..98
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 106..112
FT /evidence="ECO:0007829|PDB:1SX5"
FT TURN 113..115
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 116..118
FT /evidence="ECO:0007829|PDB:1EOP"
FT STRAND 119..123
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 125..127
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 128..140
FT /evidence="ECO:0007829|PDB:1SX5"
FT TURN 145..148
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 153..158
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 162..172
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 173..176
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 177..183
FT /evidence="ECO:0007829|PDB:1B94"
FT TURN 184..187
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 188..191
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 196..201
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 209..217
FT /evidence="ECO:0007829|PDB:1SX5"
FT HELIX 223..226
FT /evidence="ECO:0007829|PDB:1SX5"
FT STRAND 227..229
FT /evidence="ECO:0007829|PDB:1B95"
FT HELIX 233..241
FT /evidence="ECO:0007829|PDB:1SX5"
SQ SEQUENCE 245 AA; 28650 MW; 7DB7EC640A746281 CRC64;
MSLRSDLINA LYDENQKYDV CGIISAEGKI YPLGSDTKVL STIFELFSRP IINKIAEKHG
YIVEEPKQQN HYPDFTLYKP SEPNKKIAID IKTTYTNKEN EKIKFTLGGY TSFIRNNTKN
IVYPFDQYIA HWIIGYVYTR VATRKSSLKT YNINELNEIP KPYKGVKVFL QDKWVIAGDL
AGSGNTTNIG SIHAHYKDFV EGKGIFDSED EFLDYWRNYE RTSQLRNDKY NNISEYRNWI
YRGRK
