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PDBsum entry 3kx5

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protein ligands Protein-protein interface(s) links
Oxidoreductase PDB id
3kx5
Jmol
Contents
Protein chains
455 a.a. *
Ligands
HEM ×2
SO4
Waters ×1212
* Residue conservation analysis
PDB id:
3kx5
Name: Oxidoreductase
Title: Crystal structure of bacillus megaterium bm3 heme domain mut
Structure: Bifunctional p-450/NADPH-p450 reductase. Chain: a, b. Fragment: heme domain (unp residues 2-471). Synonym: flavocytochrome p450 bm3, cytochrome p450(bm-3), p cytochrome p450 102, NADPH--cytochrome p450 reductase. Engineered: yes. Mutation: yes
Source: Bacillus megaterium. Organism_taxid: 1404. Gene: cyp102a1, cyp102. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
1.69Å     R-factor:   0.175     R-free:   0.208
Authors: H.M.Girvan,C.W.Levy,D.Leys,A.W.Munro
Key ref: H.M.Girvan et al. (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. PubMed id: 20180779
Date:
02-Dec-09     Release date:   19-May-10    
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.
455 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    
 
 
Biochem J 427:455-466 (2010)
PubMed id: 20180779  
 
 
Glutamate-haem ester bond formation is disfavoured in flavocytochrome P450 BM3: characterization of glutamate substitution mutants at the haem site of P450 BM3.
H.M.Girvan, C.W.Levy, P.Williams, K.Fisher, M.R.Cheesman, S.E.Rigby, D.Leys, A.W.Munro.
 
  ABSTRACT  
 
Bacillus megaterium flavocytochrome P450 BM3 (CYP102A1) is a biotechnologically important cytochrome P450/P450 reductase fusion enzyme. Mutants I401E, F261E and L86E were engineered near the haem 5-methyl group, to explore the ability of the glutamate carboxylates to form ester linkages with the methyl group, as observed for eukaryotic CYP4 relatives. Although no covalent linkage was detected, mutants displayed marked alterations in substrate/inhibitor affinity, with L86E and I401E mutants having lower Kd values for arachidonic acid and dodecanoic (lauric) acid than WT (wild-type) BM3. All mutations induced positive shifts in haem Fe(III)/Fe(II) potential, with substrate-free I401E (-219 mV) being >170 mV more positive than WT BM3. The elevated potential stimulated FMN-to-haem electron transfer ~2-fold (to 473 s-1) in I401E, and resulted in stabilization of Fe(II)O2 complexes in the I401E and L86E P450s. EPR demonstrated some iron co-ordination by glutamate carboxylate in L86E and F261E mutants, indicating structural plasticity in the haem domains. The Fe(II)O2 complex is EPR-silent, probably resulting from antiferromagnetic coupling between Fe(III) and bound superoxide in a ferric superoxo species. Structural analysis of mutant haem domains revealed modest rearrangements, including altered haem propionate interactions that may underlie the thermodynamic perturbations observed. The mutant flavocytochromes demonstrated WT-like hydroxylation of dodecanoic acid, but regioselectivity was skewed towards omega-3 hydroxydodecanoate formation in F261E and towards omega-1 hydroxydodecanoate production in I401E. Our data point strongly to a likelihood that glutamate-haem linkages are disfavoured in this most catalytically efficient P450, possibly due to the absence of a methylene radical species during catalysis.
 

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.  
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