spacer
spacer

PDBsum entry 1d5n

Go to PDB code: 
protein metals Protein-protein interface(s) links
Oxidoreductase PDB id
1d5n
Jmol
Contents
Protein chains
205 a.a. *
Metals
_MN ×4
Waters ×1085
* Residue conservation analysis
PDB id:
1d5n
Name: Oxidoreductase
Title: Crystal structure of e. Coli mnsod at 100k
Structure: Protein (manganese superoxide dismutase). Chain: a, b, c, d. Ec: 1.15.1.1
Source: Escherichia coli. Organism_taxid: 562
Biol. unit: Dimer (from PQS)
Resolution:
1.55Å     R-factor:   0.194     R-free:   0.233
Authors: G.E.O.Borgstahl,M.Pokross,R.Chehab,A.Sekher,E.H.Snell
Key ref:
G.E.Borgstahl et al. (2000). Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase. J Mol Biol, 296, 951-959. PubMed id: 10686094 DOI: 10.1006/jmbi.1999.3506
Date:
08-Oct-99     Release date:   02-Mar-00    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P00448  (SODM_ECOLI) -  Superoxide dismutase [Mn]
Seq:
Struc:
206 a.a.
205 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.1.15.1.1  - Superoxide dismutase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: 2 superoxide + 2 H+ = O2 + H2O2
2 × superoxide
+ 2 × H(+)
= O(2)
+ H(2)O(2)
      Cofactor: Fe cation or Mn(2+) or (Zn(2+) and Cu cation)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   1 term 
  Biological process     cellular response to selenium ion   7 terms 
  Biochemical function     antioxidant activity     6 terms  

 

 
    Added reference    
 
 
DOI no: 10.1006/jmbi.1999.3506 J Mol Biol 296:951-959 (2000)
PubMed id: 10686094  
 
 
Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase.
G.E.Borgstahl, M.Pokross, R.Chehab, A.Sekher, E.H.Snell.
 
  ABSTRACT  
 
Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD). Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K. Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand. The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions. The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Diagrams of the five-coordinate trigonal bipyramidal and six-coordinate octahedral active sites of E. coli MnSOD. For overproduction of MnSOD, the E. coli strain OX326A.1 (pTTQA10) was used. This strain lacks endogenous Mn and FeSODs and harbors an ampicillin-resistant, IPTG-inducible expression plasmid for E. coli MnSOD [Hopkin et al 1992 and Steinman 1992]. Bacterial cultures were grown at 37 °C in four liters of Terrific Broth supplemented with 4 mg MnSO[4] and 200 mg ampicillin in a VRTI-culture fermentor (10 l O[2]/minute, 300 RPM). At mid-log stage the culture was induced with 1 mM IPTG. After four hours, cells were harvested, resuspended in 50 mM potassium phosphate (pH 7.5), lysed by sonication and centrifuged. The following rapid purification protocol was adapted from published procedures [Beck et al 1988, Borgstahl et al 1992 and Keele et al 1970]. The clarified lysate was heated to 60 °C for one hour and centrifuged. The supernatant was then dialyzed against 5 mM potassium phosphate (pH 7.5), mixed with preequilibrated DE-52 resin and incubated for one hour. The filtrate was then dialyzed against 2.5 mM MES (pH 5.5) and applied to a PerSeptive BioSystems CM/M POROS® column. The column was thoroughly washed and eluted with a gradient of 2.5 mM to 500 mM MES (pH 5.5). Very pure MnSOD eluted at approximately 200 mM MES. Peak fractions were pooled and dialyzed against 20 mM potassium phosphate (pH 7.0). Approximately 15-20 mg of MnSOD was purified per liter of culture. Activity was monitored with a qualitative native polyacrylamide gel electrophoresis based assay stained with p-nitroblue tetrazolium (Calbiochem). Protein samples were concentrated to 20 mg/ml and crystallized by the vapor diffusion method using sitting drops containing 2 µl of protein at 15 mg/ml plus 2 µl of reservoir solution. Drops were equilibrated over 500 µl of reservoir solution and crystals appeared in seven days. A long single crystal was grown from 50 mM bicine (pH 8.5) and 25 % PEG 6000. This crystal was square in cross section (200 µm×200 µm), over 1 mm long and was cut into several pieces each approximately 250 µm long. Pieces 2 and 3 were cryocooled and diffraction data collected at SSRL beamline 7-1 for structure determination. The structure was determined using data from piece 3. Piece 1 was mounted in a capillary and room temperature crystal mosaicity measurements were taken at SSRL beamline 1-5 in unfocussed mode using super-fine phi, Greek slicing the following day. Mosaicity values of the order of 0.008 ° were measured.
Figure 2.
Figure 2. Stereopairs of ribbon diagrams of the MnSOD structure. (a) The homodimer; monomers are colored cyan and green, and the active site manganese atom is indicated with a pink sphere. (b) An individual monomer with pink manganese and six-coordinate active site. The N-terminal a-helical domain is colored purple and the C-terminal a/b domain is colored with cyan a-helices and yellow antiparallel b-pleated sheets. (c) Close-up view of the six-coordinate active site including the manganese ligands as well as the residues that neighbor the two hydroxide ligands.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 296, 951-959) copyright 2000.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21182595 T.Nakamura, K.Torikai, K.Uegaki, J.Morita, K.Machida, A.Suzuki, and Y.Kawata (2011).
Crystal structure of the cambialistic superoxide dismutase from Aeropyrum pernix K1--insights into the enzyme mechanism and stability.
  FEBS J, 278, 598-609.
PDB codes: 3ak1 3ak2 3ak3
20972560 H.Xiang, G.Pan, C.R.Vossbrinck, R.Zhang, J.Xu, T.Li, Z.Zhou, C.Lu, and Z.Xiang (2010).
A Tandem Duplication of Manganese Superoxide Dismutase in Nosema bombycis and Its Evolutionary Origins.
  J Mol Evol, 71, 401-414.  
19265433 J.J.Perry, A.S.Hearn, D.E.Cabelli, H.S.Nick, J.A.Tainer, and D.N.Silverman (2009).
Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis.
  Biochemistry, 48, 3417-3424.
PDB codes: 1zsp 1zte 1zuq 2p4k
19505123 L.C.Tabares, J.Gätjens, C.Hureau, M.R.Burrell, L.Bowater, V.L.Pecoraro, S.Bornemann, and S.Un (2009).
pH-dependent structures of the manganese binding sites in oxalate decarboxylase as revealed by high-field electron paramagnetic resonance.
  J Phys Chem B, 113, 9016-9025.  
16999822 K.Chockalingam, J.Luba, H.S.Nick, D.N.Silverman, and H.Zhao (2006).
Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition.
  FEBS J, 273, 4853-4861.  
  16582477 R.J.Dennis, E.Micossi, J.McCarthy, E.Moe, E.J.Gordon, S.Kozielski-Stuhrmann, G.A.Leonard, and S.McSweeney (2006).
Structure of the manganese superoxide dismutase from Deinococcus radiodurans in two crystal forms.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 62, 325-329.
PDB codes: 2cdy 2ce4
  16511113 I.W.Boucher, A.K.Kalliomaa, V.M.Levdikov, E.V.Blagova, M.J.Fogg, J.A.Brannigan, K.S.Wilson, and A.J.Wilkinson (2005).
Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 61, 621-624.
PDB codes: 1xre 1xuq
15062777 A.F.Miller (2004).
Superoxide dismutases: active sites that save, but a protein that kills.
  Curr Opin Chem Biol, 8, 162-168.  
12554961 A.Vahedi-Faridi, J.Porta, and G.E.Borgstahl (2003).
Improved three-dimensional growth of manganese superoxide dismutase crystals on the International Space Station.
  Acta Crystallogr D Biol Crystallogr, 59, 385-388.  
12056897 R.Anand, P.C.Dorrestein, C.Kinsland, T.P.Begley, and S.E.Ealick (2002).
Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution.
  Biochemistry, 41, 7659-7669.
PDB codes: 1j58 1l3j
12392545 T.Hunter, J.V.Bannister, and G.J.Hunter (2002).
Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.
  Eur J Biochem, 269, 5137-5148.  
11053832 C.J.Bond, J.Huang, R.Hajduk, K.E.Flick, P.J.Heath, and B.L.Stoddard (2000).
Cloning, sequence and crystallographic structure of recombinant iron superoxide dismutase from Pseudomonas ovalis.
  Acta Crystallogr D Biol Crystallogr, 56, 1359-1366.
PDB code: 1dt0
10944335 H.D.Bellamy, E.H.Snell, J.Lovelace, M.Pokross, and G.E.Borgstahl (2000).
The high-mosaicity illusion: revealing the true physical characteristics of macromolecular crystals.
  Acta Crystallogr D Biol Crystallogr, 56, 986-995.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.