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PDBsum entry 1krj

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protein ligands metals links
Oxidoreductase PDB id
1krj
Jmol
Contents
Protein chain
294 a.a. *
Ligands
HEM
Metals
__K
Waters ×599
* Residue conservation analysis
PDB id:
1krj
Name: Oxidoreductase
Title: Engineering calcium-binding site into cytochromE C peroxidase (ccp)
Structure: CytochromE C peroxidase. Chain: a. Fragment: proximal cation-binding loop. Synonym: ccp. Engineered: yes. Mutation: yes
Source: Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: opbyc. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Resolution:
2.00Å     R-factor:   0.192    
Authors: C.A.Bonagura,B.Bhaskar,M.Sundaramoorthy,T.L.Poulos
Key ref:
C.A.Bonagura et al. (1999). Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase. J Biol Chem, 274, 37827-37833. PubMed id: 10608846 DOI: 10.1074/jbc.274.53.37827
Date:
09-Jan-02     Release date:   23-Jan-02    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00431  (CCPR_YEAST) -  Cytochrome c peroxidase, mitochondrial
Seq:
Struc:
361 a.a.
294 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 5 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.1.11.1.5  - Cytochrome-c peroxidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: 2 ferrocytochrome c + H2O2 = 2 ferricytochrome c + 2 H2O
2 × ferrocytochrome c
+ H(2)O(2)
= 2 × ferricytochrome c
+ 2 × H(2)O
      Cofactor: Heme
Heme
Bound ligand (Het Group name = HEM) matches with 95.00% similarity
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   2 terms 
  Biochemical function     peroxidase activity     2 terms  

 

 
    reference    
 
 
DOI no: 10.1074/jbc.274.53.37827 J Biol Chem 274:37827-37833 (1999)
PubMed id: 10608846  
 
 
Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase.
C.A.Bonagura, B.Bhaskar, M.Sundaramoorthy, T.L.Poulos.
 
  ABSTRACT  
 
We have previously shown that the K(+) site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A. , Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107-6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538-5545). All other peroxidases bind Ca(2+) rather than K(+). Using the K(+)-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca(2+) selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na(+), is bound instead of Ca(2+). This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca(2+) binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp(191) radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca(2+) more effectively than K(+), demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.
 
  Selected figure(s)  
 
Figure 1.
Fig. 1. Schematic model of CCP showing the location of the engineered cation site. The dark loop represents residues 192-199, which constitute the cation-binding loop. Trp191 is ~8 Å from the cation.
Figure 4.
Fig. 4. Stereoscopic models of the cation-binding sites in engineered CCP mutants, ascorbate peroxidase, and lignin peroxidase. APX, ascorbate peroxidase; LIP, lignin peroxidase.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (1999, 274, 37827-37833) copyright 1999.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20460380 Y.Ono, K.Ojima, F.Torii, E.Takaya, N.Doi, K.Nakagawa, S.Hata, K.Abe, and H.Sorimachi (2010).
Skeletal muscle-specific calpain is an intracellular Na+-dependent protease.
  J Biol Chem, 285, 22986-22998.  
19388706 J.M.Pauff, and R.Hille (2009).
Inhibition studies of bovine xanthine oxidase by luteolin, silibinin, quercetin, and curcumin.
  J Nat Prod, 72, 725-731.  
12706559 Y.Lu, J.Liu, J.Li, P.J.Bruesehoff, C.M.Pavot, and A.K.Brown (2003).
New highly sensitive and selective catalytic DNA biosensors for metal ions.
  Biosens Bioelectron, 18, 529-540.  
10981626 F.Cedrone, A.Ménez, and E.Quéméneur (2000).
Tailoring new enzyme functions by rational redesign.
  Curr Opin Struct Biol, 10, 405-410.  
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.