EzCatDB: S00403
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DB codeS00403
RLCP classification1.15.9400.1180 : Hydrolysis
CATH domainDomain 13.40.580.10 : ECO RI Endonuclease; Chain ACatalytic domain
E.C.3.1.21.4


Enzyme Name
UniProtKBKEGG

P00642P43642
Protein nameType-2 restriction enzyme EcoRIType-2 restriction enzyme MunItype II site-specific deoxyribonuclease
type II restriction enzyme
SynonymsR.EcoRI
EC 3.1.21.4
Type II restriction enzyme EcoRI
Endonuclease EcoRI
R.MunI
EC 3.1.21.4
Type II restriction enzyme MunI
Endonuclease MunI
PfamPF02963 (EcoRI)
[Graphical view]
PF11407 (RestrictionMunI)
[Graphical view]


UniProtKB:Accession NumberP00642P43642
Entry nameT2E1_ECOLXT2MU_MYCSP
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
SubunitHomodimer.
Subcellular location

CofactorBinds 2 magnesium ions per subunit.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProducts
KEGG-idC00305C00039C00001C00578C00039
CompoundMagnesiumDNAH2ODNA 5'-phosphateDNA
Typedivalent metal (Ca2+, Mg2+)nucleic acidsH2Onucleic acids,phosphate group/phosphate ionnucleic acids
ChEBI18420

15377


PubChem888

962
22247451


             
1eriAUnboundAnalogue:T-C-G-C-G-A-A-T-T-C-G-C-G(chain B) UnboundUnbound
1qc9AUnboundUnbound UnboundUnbound
1qc9BUnboundUnbound UnboundUnbound
1qc9CUnboundUnbound UnboundUnbound
1ckqAUnboundBound:T-C-G-C-G-A-A-T-T-C-G-C-G(chain B) UnboundUnbound
1cl8AUnboundBound:T-C-G-C-G-A-PRN-T-T-C-G-C-G(chain B) UnboundUnbound
1qpsAAnalogue:_MNUnbound Bound:A-A-T-T-C-G-C-G(chain N)Bound:T-C-G-C-G(chain M)
1qrhAUnboundBound:T-C-G-C-G-A-A-T-T-C-G-C-G(chain M) UnboundUnbound
1qriAUnboundBound:T-C-G-C-G-A-A-T-T-C-G-C-G(chain M) UnboundUnbound
1d02AUnboundBound:G-C-C-A-A-T-T-G-G-C(chain D:double stranded DNA) UnboundUnbound
1d02BUnboundBound:G-C-C-A-A-T-T-G-G-C(chain C:double stranded DNA) UnboundUnbound

Active-site residues
resource
Swiss-prot;P00624
pdbCatalytic residuesCofactor-binding residuescomment
           
1eriALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1qc9ALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1qc9BLYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1qc9CLYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1ckqALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1cl8ALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1qpsALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1qrhALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1qriALYS 113
ASP 91;GLU 111(two Mg2+ binding)
 
1d02ALYS 100
      ;GLU  98(two Mg2+ binding)
mutant D83A
1d02BLYS 100
      ;GLU  98(two Mg2+ binding)
mutant D83A

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[6]Fig.8, Fig.11, p.12-172
[7]Fig.5, p.13492-134942
[8]p.5810-5811
[9]Fig1, p.6

references
[1]
CommentsX-ray crystallography (3 Angstroms)
Medline ID87069951
PubMed ID3024321
JournalScience
Year1986
Volume234
Pages1526-41
AuthorsMcClarin JA, Frederick CA, Wang BC, Greene P, Boyer HW, Grable J, Rosenberg JM
TitleStructure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.
Related UniProtKBP00642
[2]
Commentsmutation analysis
Medline ID91072344
PubMed ID2254311
JournalJ Biol Chem
Year1990
Volume265
Pages21520-6
AuthorsHager PW, Reich NO, Day JP, Coche TG, Boyer HW, Rosenberg JM, Greene PJ
TitleProbing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements.
Related UniProtKBP00642
[3]
CommentsX-ray crystallography (2.7 Angstroms)
Medline ID90378308
PubMed ID2399465
JournalScience
Year1990
Volume249
Pages1307-9
AuthorsKim YC, Grable JC, Love R, Greene PJ, Rosenberg JM
TitleRefinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.
Related UniProtKBP00642
[4]
Commentsmolecular dynamics simulations
PubMed ID7727057
JournalJ Biomol Struct Dyn
Year1994
Volume12
Pages487-525
AuthorsKumar S, Duan Y, Kollman PA, Rosenberg JM
TitleMolecular dynamics simulations suggest that the Eco RI kink is an example of molecular strain.
Related PDB1eri
[5]
Commentscatalysis
PubMed ID7607482
JournalGene
Year1995
Volume157
Pages157-62
AuthorsJeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
TitleEvidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.
[6]
Commentscatalysis
PubMed ID9210460
JournalEur J Biochem
Year1997
Volume246
Pages1-22
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
[7]
CommentsX-ray crystallography (2.15 Angstroms)
PubMed ID9811827
JournalProc Natl Acad Sci U S A
Year1998
Volume95
Pages13489-94
AuthorsHorton NC, Newberry KJ, Perona JJ
TitleMetal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
Related PDB1bss
[8]
CommentsX-ray crystallography (1.7 Angstroms)
PubMed ID10545092
JournalEMBO J
Year1999
Volume18
Pages5805-16
AuthorsDeibert M, Grazulis S, Janulaitis A, Siksnys V, Huber R
TitleCrystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.
Related PDB1d02
[9]
PubMed ID10739241
JournalProtein Sci
Year2000
Volume9
Pages1-9
AuthorsDall'Acqua W, Carter P
TitleSubstrate-assisted catalysis: molecular basis and biological significance.

comments
These enzymes, EcoRI(PDB; 1eri, 1qc9) and MunI(PDB; 1d02) belong to the type II restriction endonucleases.
According to the paper [6], cleavage of DNA by restriction endonucleases yields 3'-OH and 5'-phosphate ends, where hydrolysis of the phosphodiester bonds by EcoRI and EcoRV occurs with inversion of configuration at the phosphorous atom, suggesting an attack of a water molecule in line with the 3'-OH leaving group. In general, hydrolysis of phosphodiester bonds requires three functional entities as follows [6]:
(1) A general base that activates the attacking nucleophile,
(2) A Lewis acid that stabilizes the extra negative charge in the pentacovalent transition state,
(3) An acid that protonates or stabilizes the leaving group.
The literature [6] also described the two possible catalytic mechanisms, the substrate-assisted catalysis model and the two-metal-ion mechanism, as described in the following paragraph. However, this paper supported the substrate-assisted catalysis model more favorably than the two-metal-ion mechanism.
The substrate-assisted catalysis model: The attacking water molecule is oriented and deprotonated by the next phosphate group 3' to the scissile phosphate. The negative charge of the transition state could be stablized by the Mg2+ ion and the semi-conserved lysine. The metal ion is bound by the two conserved acidc amino acid residues. The 3'-O- leaving group is protonated by a Mg2+-bound water [6].
The two-metal-ion mechanism: A metal ion bound at one site is responsible for charge neutralization at the scissile phosphate. The attacking water is considered to be part of the hydration sphere of a metal ion bound at the second site [6].
The literature [7] suggested another possible mechanism, three-metal ion mechanism for type II restriction endonucleases from the structural data, as follows:
A metal ion at site I ligates through water to the 3'-phosphate. A second inner-sphere water molecule on this metal dissociates to provide the attacking hydroxide ion, and this dissociation is aided by the immediately adjacent Lysine, corresponding to Lys113 in EcoRI. The metal at site III provides stabilization of the incipient negative charge as the transtion state develops. An inner-sphere water on this metal is located within hydrogen-bonding distance of the leaving 3'-oxygen. Thus, the site III metal is suggested to be operative in lowering the pKa of this water, so that it may dissociate to immediately protonate the leaving anion [7]. The site II metal is purely structural [7].
Crystal structures of these type II endonucleases, EcoRV, EcoRI and PvuII bound to DNA show that the relative positions of the scissile and adjacent 3'-phosphates are conserved. Therefore, the two metal ions bound in site I and site III may have similar functions in each of these enzymes [7].
###
In the structures with DNA substrate (PDB; 1eri), acidic residues at the active site seems to be disoriented. However, the pattern of the active site structure of 1eri (PDB) is similar to those of EcoRV, BglI and PvuII (S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes.
###
More recently, several papers including [9] supported the substrate-assisted mechanism for this enzyme and related enzymes (type II restriction enzymes), ruling out the two-metal-ion mechanism. Thus, we concluded that this enzyme adopts the substrate-assisted mechanism with only one metal ion for catalysis (see EcoRV; S00404 in EzCatDB).

createdupdated
2002-09-272009-02-26


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