DB code: S00384

RLCP classification 1.15.8300.1170 : Hydrolysis
CATH domain 3.40.91.20 : Restriction Endonuclease Catalytic domain
E.C. 3.1.21.4
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Pfam
P23940 Type-2 restriction enzyme BamHI
R.BamHI
EC 3.1.21.4
Type II restriction enzyme BamHI
Endonuclease BamHI
PF02923 (BamHI)
[Graphical View]

KEGG enzyme name
type II site-specific deoxyribonuclease
type II restriction enzyme

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P23940 T2BA_BACAM Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates. Homodimer. Binds 2 magnesium ions per subunit.

KEGG Pathways
Map code Pathways E.C.

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00039 C00001 C00578 C00039
E.C.
Compound Magnesium DNA H2O DNA 5'-phosphate DNA
Type divalent metal (Ca2+, Mg2+) nucleic acids H2O nucleic acids,phosphate group/phosphate ion nucleic acids
ChEBI 18420
15377
PubChem 888
22247451
962
1bamA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1bhmA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-A-T-G-G-A-T-C-C-A-T (chain D) Unbound Unbound
1bhmB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-A-T-G-G-A-T-C-C-A-T-A (chain C) Unbound Unbound
1esgA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:T-G-G-A-T-T-C-A (chain C) Unbound Unbound
1esgB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:T-G-A-A-T-C-C-A (chain D) Unbound Unbound
2bamA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:2x_CA Bound:T-A-T-G-G-A-T-C-C-A-T (chain D) Unbound Unbound
2bamB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-A-T-G-G-A-T-C-C-A-T-A (chain C) Unbound Unbound
3bamA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:2x_MN Unbound Bound:G-A-T-C-C-A-T (chain E) Bound:T-A-T-G (chain D)
3bamB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-A-T-G-G-A-T-C-C-A-T-A (chain C) Unbound Unbound

Reference for Active-site residues
resource references E.C.
literature [2],[6]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1bamA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1bhmA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1bhmB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1esgA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1esgB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
2bamA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
2bamB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
3bamA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)
3bamB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 113 GLU 77;ASP 94;GLU 111(two Mg2+ binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[3]
p.662
[5]
Fig.8, Fig.11, p.12-17 2
[6]
Fig.4(b), p.913 2

References
[1]
Resource
Comments X-ray crystallography (1.95 Angstroms)
Medline ID 94195433
PubMed ID 8145855
Journal Nature
Year 1994
Volume 368
Pages 660-4
Authors Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
Title Structure of restriction endonuclease BamHI and its relationship to EcoRI.
Related PDB
Related UniProtKB P23940
[2]
Resource
Comments X-ray crystallography (1.95 Angstroms)
Medline ID
PubMed ID 8081758
Journal Structure
Year 1994
Volume 2
Pages 439-52
Authors Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
Title Structure of restriction endonuclease bamhi phased at 1.95 A resolution by MAD analysis.
Related PDB 1bam
Related UniProtKB
[3]
Resource
Comments X-ray crystallography (2.2 Angstroms)
Medline ID
PubMed ID 7624794
Journal Science
Year 1995
Volume 269
Pages 656-63
Authors Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
Title Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding.
Related PDB 1bhm
Related UniProtKB
[4]
Resource
Comments catalysis
Medline ID
PubMed ID 7607482
Journal Gene
Year 1995
Volume 157
Pages 157-62
Authors Jeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
Title Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.
Related PDB
Related UniProtKB
[5]
Resource
Comments catalysis
Medline ID
PubMed ID 9210460
Journal Eur J Biochem
Year 1997
Volume 246
Pages 1-22
Authors Pingoud A, Jeltsch A
Title Recognition and cleavage of DNA by type-II restriction endonucleases.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-ray crystallography (1.8 Angstroms)
Medline ID 98455073
PubMed ID 9783752
Journal Nat Struct Biol
Year 1998
Volume 5
Pages 910-6
Authors Viadiu H, Aggarwal AK
Title The role of metals in catalysis by the restriction endonuclease BamHI.
Related PDB 2bam 3bam
Related UniProtKB P23940
[7]
Resource
Comments X-ray crystallography (2.15 Angstroms)
Medline ID
PubMed ID 9811827
Journal Proc Natl Acad Sci U S A
Year 1998
Volume 95
Pages 13489-94
Authors Horton NC, Newberry KJ, Perona JJ
Title Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
Related PDB
Related UniProtKB
[8]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 10882125
Journal Mol Cell
Year 2000
Volume 5
Pages 889-95
Authors Viadiu H, Aggarwal AK
Title Structure of BamHI bound to nonspecific DNA: a model for DNA sliding.
Related PDB 1esg
Related UniProtKB

Comments
This enzyme belongs to the type II restriction endonucleases.
According to the paper [5], 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 [5]:
(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 [5] 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 [5].
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 [5].
The literature [6] proposed a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of other endonucleases such as EcoRV, instead, similar to that proposed for the 3'-5'-exonuclease domain of the E. coli DNA polymerase I (see M00055-[8]).
BamHI is the only restriction endonuclease to be characterized structurally that has a glutamic acid (Glu113) in the position corresponding to a lysine in the others [6]. Glu113 appears to act as a base in accepting a proton from the attacking water [6].
A metal-bound water molecule has a lower pKa value (11.4 for Mg2+, 10.6 for Mn2+) than bulk water, favoring the formation of a nucleophilic hydroxyl species that can attack the scissile phosphodiester. The metal ions are in positions to stabilize the 'entering' and 'leaving' oxygens at the apical positions, and to reduce the energy in forming the 90 degree O-P-O bond angles between the apical and the equatorial oxygens [6].
The mechanism of Ca2+ inhibition is also described in the literature [6]. Firstly, pKa of a Ca2+ bound water (pKa=12.9) is higher than that for Mg2+ (pKa=11.4) or Mn2+ (pKa=10.6), which will have the effect of lowering the concentration of metal-hydroxyl ions for the nucleophilic attack on the scissile phosphodiester. Secondly, Ca2+ is a bulkier ion (radius 0.99 A) than Mg2+ (0.65 A) or Mn2+ (0.80), even compared to other ions (Co2+, Zn2+, & Cd2+). Although the two Ca2+ ions appear to be well accommodated in the ground state of the BamHI-DNA complex, this may not be the case during the transition state. The transition state may cause steric interference with the larger Ca2+ ions.
Considering structure with dna and analogous ions (Ca2+), the reaction proceeds as follows:
(1) Glu113 acts as a general base to activate a water, which is bound to magnesium ion. (This ion is bound to Glu77 and Asp94.)
(2) The activated water makes a nucleophilic attack on the phoshoryc ester of DNA.
(3) A pentacovalent transition-state is stabilized by the two magnesium ions. These ions interact with a non-bridging phosphate oxygen and the leaving 3'-oxygen.
(4) 3'-end is protonated by another water, which is bound to the other magnesium ion. (This ion is bound to Asp94 and Glu77.)

Created Updated
2002-09-27 2009-02-26