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PDBsum entry 1smi

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Oxidoreductase PDB id
1smi
Jmol
Contents
Protein chains
439 a.a. *
Ligands
HEM ×2
Waters ×492
* Residue conservation analysis
PDB id:
1smi
Name: Oxidoreductase
Title: A single mutation of p450 bm3 induces the conformational rearrangement seen upon substrate-binding in wild-type enzyme
Structure: Bifunctional p-450:nadph-p450 reductase. Chain: a, b. Fragment: cytochrome p450 102. Synonym: cytochrome p450(bm-3). P450bm-3 [includes: cytochrome p450 102, NADPH--cytochrome p450 reductase]. .Cytochrome p-450:nadph-p-450 reductase. Engineered: yes. Mutation: yes
Source: Bacillus megaterium. Organism_taxid: 1404. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.00Å     R-factor:   0.227     R-free:   0.288
Authors: M.G.Joyce,H.M.Girvan,A.W.Munro,D.Leys
Key ref:
M.G.Joyce et al. (2004). A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme. J Biol Chem, 279, 23287-23293. PubMed id: 15020590 DOI: 10.1074/jbc.M401717200
Date:
09-Mar-04     Release date:   08-Jun-04    
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.
439 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.M401717200 J Biol Chem 279:23287-23293 (2004)
PubMed id: 15020590  
 
 
A single mutation in cytochrome P450 BM3 induces the conformational rearrangement seen upon substrate binding in the wild-type enzyme.
M.G.Joyce, H.M.Girvan, A.W.Munro, D.Leys.
 
  ABSTRACT  
 
The multidomain fatty-acid hydroxylase flavocytochrome P450 BM3 has been studied as a paradigm model for eukaryotic microsomal P450 enzymes because of its homology to eukaryotic family 4 P450 enzymes and its use of a eukaryotic-like diflavin reductase redox partner. High-resolution crystal structures have led to the proposal that substrate-induced conformational changes lead to removal of water as the sixth ligand to the heme iron. Concomitant changes in the heme iron spin state and heme iron reduction potential help to trigger electron transfer from the reductase and to initiate catalysis. Surprisingly, the crystal structure of the substrate-free A264E heme domain mutant reveals the enzyme to be in the conformation observed for substrate-bound wild-type P450, but with the iron in the low-spin state. This provides strong evidence that the spin-state shift observed upon substrate binding in wild-type P450 BM3 not only is caused indirectly by structural changes in the protein, but is a direct consequence of the presence of the substrate itself, similar to what has been observed for P450cam. The crystal structure of the palmitoleate-bound A264E mutant reveals that substrate binding promotes heme ligation by Glu(264), with little other difference from the palmitoleate-bound wild-type structure observable. Despite having a protein-derived sixth heme ligand in the substrate-bound form, the A264E mutant is catalytically active, providing further indication for structural rearrangement of the active site upon reduction of the heme iron, including displacement of the glutamate ligand to allow binding of dioxygen.
 
  Selected figure(s)  
 
Figure 1.
FIG. 1. Stereo view of an overlay of the substrate-free A264E heme domain (green ribbons). Upper, substrate-free form (yellow) of the wild-type P450 BM3 heme domain; lower, substrate-bound form (blue) of the wild-type P450 BM3 heme domain.
Figure 4.
FIG. 4. Stereo view of the active site of A264E. Upper, overlay of the active site of A264E (blue) and N-palmitoylglycine-bound wild-type P450 BM3 (green) (Protein Data Bank code 1JPZ [PDB] ). For clarity, the heme macrocycle is displayed only for the A264E mutant. The pattern of hydrogen bonding to site H is indicated by the dotted lines. The ligating water molecule occupying site L in the A264E mutant is colored red. Lower, active-site structure of the palmitoleic acid-bound form of the A264E heme domain. Residues are colored according to residue type; the substrate is depicted in purple.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 23287-23293) copyright 2004.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21431175 T.N.Waltham, H.M.Girvan, C.F.Butler, S.R.Rigby, A.J.Dunford, R.A.Holt, and A.W.Munro (2011).
Analysis of the oxidation of short chain alkynes by flavocytochrome P450 BM3.
  Metallomics, 3, 369-378.  
21240430 W.C.Huang, P.M.Cullis, E.L.Raven, and G.C.Roberts (2011).
Control of the stereo-selectivity of styrene epoxidation by cytochrome P450 BM3 using structure-based mutagenesis.
  Metallomics, 3, 410-416.  
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
  21048857 I.Axarli, A.Prigipaki, and N.E.Labrou (2010).
Cytochrome P450 102A2 Catalyzes Efficient Oxidation of Sodium Dodecyl Sulphate: A Molecular Tool for Remediation.
  Enzyme Res, 2010, 125429.  
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
18392864 C.K.Chen, T.K.h.Shokhireva, R.E.Berry, H.Zhang, and F.A.Walker (2008).
The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity.
  J Biol Inorg Chem, 13, 813-824.  
17957765 E.Stjernschantz, B.M.van Vugt-Lussenburg, A.Bonifacio, S.B.de Beer, G.van der Zwan, C.Gooijer, J.N.Commandeur, N.P.Vermeulen, and C.Oostenbrink (2008).
Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3.
  Proteins, 71, 336-352.  
18483737 M.Dietrich, S.Eiben, C.Asta, T.A.Do, J.Pleiss, and V.B.Urlacher (2008).
Cloning, expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from Bacillus licheniformis.
  Appl Microbiol Biotechnol, 79, 931-940.  
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.  
16600969 M.S.Yousef, N.Bischoff, C.M.Dyer, W.A.Baase, and B.W.Matthews (2006).
Guanidinium derivatives bind preferentially and trigger long-distance conformational changes in an engineered T4 lysozyme.
  Protein Sci, 15, 853-861.
PDB codes: 2f2q 2f32 2f47
16234920 H.E.Seward, H.M.Girvan, and A.W.Munro (2005).
Cytochrome P450s: creating novel ligand sets.
  Dalton Trans, (), 3419-3426.  
15286283 M.S.Yousef, W.A.Baase, and B.W.Matthews (2004).
Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme.
  Proc Natl Acad Sci U S A, 101, 11583-11586.
PDB codes: 1t8a 1t97
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.

 

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