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PDBsum entry 2uwh

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protein ligands Protein-protein interface(s) links
Oxidoreductase PDB id
2uwh
Jmol
Contents
Protein chains
(+ 0 more) 458 a.a. *
Ligands
HEM ×6
PLM ×6
Waters ×519
* Residue conservation analysis
PDB id:
2uwh
Name: Oxidoreductase
Title: Cytochrome p450 bm3 mutant in complex with palmitic acid
Structure: Bifunctional p-450\: NADPH-p450 reductase. Chain: a, b, c, d, e, f. Fragment: heme domain, residues 1-458. Synonym: cytochrome p450bm3, cytochrome p450(bm-3), p450bm-3. Engineered: yes. Mutation: yes. Other_details: palmitate bound
Source: Bacillus megaterium. Organism_taxid: 1404. Atcc: 14581. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.80Å     R-factor:   0.219     R-free:   0.299
Authors: W.-C.Huang,M.G.Joyce,A.C.G.Westlake,G.C.K.Roberts, P.C.E.Moody
Key ref:
W.C.Huang et al. (2007). Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency. J Mol Biol, 373, 633-651. PubMed id: 17868686 DOI: 10.1016/j.jmb.2007.08.015
Date:
21-Mar-07     Release date:   28-Aug-07    
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.
458 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: 10.1016/j.jmb.2007.08.015 J Mol Biol 373:633-651 (2007)
PubMed id: 17868686  
 
 
Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency.
W.C.Huang, A.C.Westlake, J.D.Maréchal, M.G.Joyce, P.C.Moody, G.C.Roberts.
 
  ABSTRACT  
 
Cytochrome P450BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase, is a member of a very large superfamily of monooxygenase enzymes. The available crystal structures of the enzyme show non-productive binding of substrates with their omega-end distant from the iron in a hydrophobic pocket at one side of the active site. We have constructed and characterised mutants in which this pocket is filled by large hydrophobic side-chains replacing alanine at position 82. The mutants having phenylalanine or tryptophan at this position have very much (approximately 800-fold) greater affinity for substrate, with a greater conversion of the haem iron to the high-spin state, and similarly increased catalytic efficiency. The enzyme as isolated contains bound palmitate, reflecting this much higher affinity. We have determined the crystal structure of the haem domain of the Ala82Phe mutant with bound palmitate; this shows that the substrate is binding differently from the wild-type enzyme but still distant from the haem iron. Detailed analysis of the structure indicates that the tighter binding in the mutant reflects a shift in the conformational equilibrium of the substrate-free enzyme towards the conformation seen in the substrate complex rather than differences in the enzyme-substrate interactions. On this basis, we outline a sequence of events for the initial stages of the catalytic cycle. The Ala82Phe and Ala82Trp mutants are also very much more effective catalysts of indole hydroxylation than the wild-type enzyme, suggesting that they will be valuable starting points for the design of mutants to catalyse synthetically useful hydroxylation reactions.
 
  Selected figure(s)  
 
Figure 4.
Figure 4. Structure of the palmitate complex of the haem domain of the A82F mutant of P450BM3. (a) Overall structure of the palmitate complex of the A82F mutant (cyan) compared to the N-palmitoylglycine complex of the wild-type enzyme (PDB code 1JPZ; green). The haem group is shown in red, the palmitate bound to A82F in brown and the N-palmitoylglycine bound to wild-type in purple; the mutated Phe82 residue is highlighted in yellow. The F and G helices are indicated. (b) Stereo view of the active site in the crystal structure of the A82F mutant. The side-chain of the mutated Phe82 residue is highlighted in magenta. The bound palmitate is in dark green, the haem in red and Tyr51, Phe87 and Phe81 are in yellow. The blue mesh is the electron density map calculated using the omit procedure (two cycles) in SFCHECK.^57 The omit maps for all six chains in the asymmetric unit are shown in Supplementary Data. (c) Comparison of the substrate-binding site in the substrate-free and substrate bound wild-type enzyme with that in the substrate-bound A82F mutant. Relevant substrate contact residues are shown. The A82F mutant with palmitate bound (2UWH) is in pink, the wild-type with N-palmitoylglycine bound (1JPZ) is in cyan and the wild-type without substrate (2BMH) is in green.
Figure 8.
Figure 8. Docking of indole into the active site of P450 BM3. Indole is shown as a space-filling model, docked into the active site of (a) the wild-type enzyme (1JPZ) and (b) the A82F mutant (2UWH). Side-chains of F81 (yellow), F82/A82 (pink) and F87 (green) are shown.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 373, 633-651) copyright 2007.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21161210 R.K.Gudiminchi, and M.S.Smit (2011).
Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1.
  Appl Microbiol Biotechnol, 90, 117-126.  
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.  
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.  
20593075 G.B.Shul'pin (2010).
Selectivity enhancement in functionalization of C-H bonds: A review.
  Org Biomol Chem, 8, 4217-4228.  
20947800 M.E.Ener, Y.T.Lee, J.R.Winkler, H.B.Gray, and L.Cheruzel (2010).
Photooxidation of cytochrome P450-BM3.
  Proc Natl Acad Sci U S A, 107, 18783-18786.  
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.  
19492389 C.J.Whitehouse, S.G.Bell, W.Yang, J.A.Yorke, C.F.Blanford, A.J.Strong, E.J.Morse, M.Bartlam, Z.Rao, and L.L.Wong (2009).
A highly active single-mutation variant of P450BM3 (CYP102A1).
  Chembiochem, 10, 1654-1656.
PDB code: 3hf2
19555717 I.G.Denisov, D.J.Frank, and S.G.Sligar (2009).
Cooperative properties of cytochromes P450.
  Pharmacol Ther, 124, 151-167.  
19490105 O.Nikolaeva, Y.Takahashi, G.Moiseyev, and J.X.Ma (2009).
Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane.
  FEBS J, 276, 3020-3030.  
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 code is shown on the right.