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PDBsum entry 4kf0

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protein ligands Protein-protein interface(s) links
Oxidoreductase PDB id
4kf0
Jmol
Contents
Protein chains
451 a.a.
Ligands
HEM ×2
Waters ×1036
PDB id:
4kf0
Name: Oxidoreductase
Title: Structure of the a82f p450 bm3 heme domain
Structure: Bifunctional p-450/NADPH-p450 reductase. Chain: a, b. Fragment: p450 bm3 heme domain. Synonym: cytochrome p450(bm-3), cytochrome p450bm-3, cytoch 102, NADPH--cytochrome p450 reductase. Engineered: yes. Mutation: yes
Source: Bacillus megaterium. Organism_taxid: 1404. Gene: cyp102a1, cyp102
Resolution:
1.45Å     R-factor:   0.146     R-free:   0.178
Authors: D.Leys
Key ref: C.F.Butler et al. (2013). Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias. J Biol Chem, 288, 25387-25399. PubMed id: 23828198 DOI: 10.1074/jbc.M113.479717
Date:
26-Apr-13     Release date:   10-Jul-13    
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.
451 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.M113.479717 J Biol Chem 288:25387-25399 (2013)
PubMed id: 23828198  
 
 
Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias.
C.F.Butler, C.Peet, A.E.Mason, M.W.Voice, D.Leys, A.W.Munro.
 
  ABSTRACT  
 
Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such "gatekeeper" mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme.