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PDBsum entry 2ij3

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protein ligands Protein-protein interface(s) links
Oxidoreductase PDB id
2ij3
Jmol
Contents
Protein chains
453 a.a. *
Ligands
HEM ×2
Waters ×852
* Residue conservation analysis
PDB id:
2ij3
Name: Oxidoreductase
Title: Structure of the a264h mutant of cytochrome p450 bm3
Structure: Cytochrome p450 bm3. Chain: a, b. Fragment: residues 1-470. Engineered: yes. Mutation: yes
Source: Bacillus megaterium. Organism_taxid: 1404. Gene: cyp102a1
Resolution:
1.90Å     R-factor:   0.179     R-free:   0.218
Authors: H.S.Toogood,D.Leys
Key ref:
H.M.Girvan et al. (2007). Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3. J Biol Chem, 282, 564-572. PubMed id: 17077084 DOI: 10.1074/jbc.M607949200
Date:
29-Sep-06     Release date:   07-Nov-06    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P14779  (CPXB_BACME) -  Bifunctional P-450/NADPH-P450 reductase
Seq:
Struc:
 
Seq:
Struc:
 
Seq:
Struc:
1049 a.a.
453 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class 2: E.C.1.14.14.1  - Unspecific monooxygenase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: RH + reduced flavoprotein + O2 = ROH + oxidized flavoprotein + H2O
RH
+ reduced flavoprotein
+ O(2)
= ROH
+ oxidized flavoprotein
+ H(2)O
      Cofactor: Heme-thiolate
   Enzyme class 3: E.C.1.6.2.4  - NADPH--hemoprotein reductase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: NADPH + n oxidized hemoprotein = NADP+ + n reduced hemoprotein
NADPH
+ n oxidized hemoprotein
= NADP(+)
+ n reduced hemoprotein
      Cofactor: FAD; FMN
FAD
FMN
Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     oxidation-reduction process   1 term 
  Biochemical function     oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen     3 terms  

 

 
    reference    
 
 
DOI no: 10.1074/jbc.M607949200 J Biol Chem 282:564-572 (2007)
PubMed id: 17077084  
 
 
Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3.
H.M.Girvan, H.E.Seward, H.S.Toogood, M.R.Cheesman, D.Leys, A.W.Munro.
 
  ABSTRACT  
 
Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.
 
  Selected figure(s)  
 
Figure 4.
Structures of P450 BM3 A264K/H mutants. A, heme iron coordination by Lys-264 in the SB conformation for A264K. B, His-264 heme iron coordination in the SF conformation for A264H. C, overlay of I helices and residue Phe-87 in A264K (green) and A264H (blue) heme domains. A profound distortion of the I helix and relocation of Phe-87 occurs between the SB and SF forms.
Figure 5.
A, overlay of the three distinct P450 BM3 heme domain conformations. For clarity, only those structural elements that undergo significant repositioning are depicted for all three conformations and colored green, blue, and magenta for SF2, SF1, and SB conformations, respectively. B-D, solvent-accessible surface for SF2 (B), SF1 (C), and SB (D) conformations, with N-palmitoylglycine substrate (on the basis of the position occupied in PDB 1JPZ) depicted in yellow spheres, indicating position of the active site access channel. Color coding is as in A.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 564-572) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21110374 C.J.Whitehouse, W.Yang, J.A.Yorke, B.C.Rowlatt, A.J.Strong, C.F.Blanford, S.G.Bell, M.Bartlam, L.L.Wong, and Z.Rao (2010).
Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3).
  Chembiochem, 11, 2549-2556.  
20180779 H.M.Girvan, C.W.Levy, P.Williams, K.Fisher, M.R.Cheesman, S.E.Rigby, D.Leys, and A.W.Munro (2010).
Glutamate-haem ester bond formation is disfavoured in flavocytochrome P450 BM3: characterization of glutamate substitution mutants at the haem site of P450 BM3.
  Biochem J, 427, 455-466.
PDB codes: 3kx3 3kx4 3kx5
20446763 T.C.Pochapsky, S.Kazanis, and M.Dang (2010).
Conformational plasticity and structure/function relationships in cytochromes P450.
  Antioxid Redox Signal, 13, 1273-1296.  
19074393 L.H.Xu, S.Fushinobu, H.Ikeda, T.Wakagi, and H.Shoun (2009).
Crystal structures of cytochrome P450 105P1 from Streptomyces avermitilis: conformational flexibility and histidine ligation state.
  J Bacteriol, 191, 1211-1219.
PDB codes: 3e5j 3e5k 3e5l
17534532 A.W.Munro, H.M.Girvan, and K.J.McLean (2007).
Variations on a (t)heme--novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily.
  Nat Prod Rep, 24, 585-609.  
18028029 K.J.McLean, H.M.Girvan, and A.W.Munro (2007).
Cytochrome P450/redox partner fusion enzymes: biotechnological and toxicological prospects.
  Expert Opin Drug Metab Toxicol, 3, 847-863.  
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.