spacer
spacer

PDBsum entry 2c6h
Go to PDB code: 
protein ligands links
Oxidoreductase PDB id
2c6h

 

 

 

 

Loading ...

 
JSmol PyMol  
Contents
Protein chains
394 a.a. *
Ligands
HEM ×2
PXI ×2
SO4 ×5
Waters ×216
* Residue conservation analysis
PDB id:
2c6h
Name: Oxidoreductase
Title: Crystal structure of yc-17-bound cytochrome p450 pikc (cyp107l1)
Structure: Cytochrome p450 monooxygenase. Chain: a, b. Synonym: cytochrome p450 pikc cyp107l1, cytochrome p450 hydroxylase pikc. Engineered: yes
Source: Streptomyces venezuelae. Organism_taxid: 54571. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.35Å     R-factor:   0.195     R-free:   0.231
Authors: D.H.Sherman,S.Li,L.V.Yermalitskaya,Y.Kim,J.A.Smith,M.R.Waterman, L.M.Podust
Key ref:
D.H.Sherman et al. (2006). The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J Biol Chem, 281, 26289-26297. PubMed id: 16825192 DOI: 10.1074/jbc.M605478200
Date:
09-Nov-05     Release date:   03-Jul-06    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
O87605  (PIKC_STRVZ) -  Cytochrome P450 monooxygenase PikC from Streptomyces venezuelae
Seq:
Struc: 		416 a.a.
394 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.1.14.15.33  - pikromycin synthase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction:
1. narbomycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H+ = pikromycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
2. narbomycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H+ = neopikromycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
3. narbomycin + 4 reduced [2Fe-2S]-[ferredoxin] + 2 O2 + 4 H+ = novapikromycin + 4 oxidized [2Fe-2S]-[ferredoxin] + 2 H2O
4. 10-deoxymethymycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H+ = methymycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
5. 10-deoxymethymycin + 2 reduced [2Fe-2S]-[ferredoxin] + O2 + 2 H+ = neomethymycin + 2 oxidized [2Fe-2S]-[ferredoxin] + H2O
6. 10-deoxymethymycin +
narbomycin
 + 2 × reduced [2Fe-2S]-[ferredoxin]
Bound ligand (Het Group name = PXI)
matches with 88.89% similarity 		+ O2
 + 2 × H(+)
 = pikromycin
 + 2 × oxidized [2Fe-2S]-[ferredoxin]
 + H2O
narbomycin
 + 2 × reduced [2Fe-2S]-[ferredoxin]
Bound ligand (Het Group name = PXI)
matches with 88.89% similarity 		+ O2
 + 2 × H(+)
 = neopikromycin
 + 2 × oxidized [2Fe-2S]-[ferredoxin]
 + H2O
narbomycin
 + 4 × reduced [2Fe-2S]-[ferredoxin]
Bound ligand (Het Group name = PXI)
matches with 88.89% similarity 		+ 2 × O2
 + 4 × H(+)
 = novapikromycin
 + 4 × oxidized [2Fe-2S]-[ferredoxin]
 + 2 × H2O
10-deoxymethymycin
Bound ligand (Het Group name = PXI)
corresponds exactly 		+ 2 × reduced [2Fe-2S]-[ferredoxin]
 + O2
 + 2 × H(+)
 = methymycin
 + 2 × oxidized [2Fe-2S]-[ferredoxin]
 + H2O
10-deoxymethymycin
Bound ligand (Het Group name = PXI)
corresponds exactly 		+ 2 × reduced [2Fe-2S]-[ferredoxin]
 + O2
 + 2 × H(+)
 = neomethymycin
 + 2 × oxidized [2Fe-2S]-[ferredoxin]
 + H2O
10-deoxymethymycin
Bound ligand (Het Group name = PXI)
corresponds exactly 		+
      Cofactor: Heme-thiolate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 

 
    reference    
 
 
DOI no: 10.1074/jbc.M605478200 J Biol Chem 281:26289-26297 (2006)
PubMed id: 16825192  
 
 
The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae.
D.H.Sherman, S.Li, L.V.Yermalitskaya, Y.Kim, J.A.Smith, M.R.Waterman, L.M.Podust.
 
  ABSTRACT  
 
The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 A) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 A)or narbomycin (resolution 1.7 A). A central feature of the enzyme-substrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3' dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.
 
  Selected figure(s)  
 
Figure 2.
FIGURE 2. Ribbon representation of ligand-free PikC. A, open, and B, closed conformations of ligand-free PikC (2BVJ). C, overlay of both conformations, open (cyan) and closed (gray), demonstrating that in the open form, the F and G helix are bent away from the heme to enable substrate access to the active site. Molecules in C are rotated 90° toward the viewer along a horizontal axis in the plane of drawing when compared with A and B. The F helix is not seen in this orientation. Closed conformation is related within r.m.s. deviations of 0.58 Å for C atoms to catalytically relevant YC-17- and narbomycin-bound forms. The heme co-factor is shown in red. 		Figure 6.
FIGURE 6. Functional activity of PikC mutants. High pressure liquid chromatography analyses of PikC-catalyzed reactions using YC-17 (A series) and narbomycin (B series) as substrate are shown. A1/B1, negative control in the absence of PikC. A2/B2, PikC wild type (PikC-wt). Mutants are used as indicated in the figure. Compound identities are as follows: 1, YC-17; 2, neomethymycin; 3, methymycin; 4, narbomycin; 5, pikromycin. Conversion of narbomycin at low efficiency is probably due to use of exogenous redox partners (e.g. spinach ferredoxin reductase) in P450 reconstitution assays.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 26289-26297) copyright 2006.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21154803 M.Ma, S.G.Bell, W.Yang, Y.Hao, N.H.Rees, M.Bartlam, W.Zhou, L.L.Wong, and Z.Rao (2011).
Structural Analysis of CYP101C1 from Novosphingobium aromaticivorans DSM12444.
  Chembiochem, 12, 88-99.
PDB codes: 3oft 3ofu
  21307966 P.R.Markwick, L.C.Pierce, D.B.Goodin, and J.A.McCammon (2011).
Adaptive Accelerated Molecular Dynamics (Ad-AMD) Revealing the Molecular Plasticity of P450cam.
  J Phys Chem Lett, 2, 158-164.  
21171581 Y.T.Lee, E.C.Glazer, R.F.Wilson, C.D.Stout, and D.B.Goodin (2011).
Three clusters of conformational States in p450cam reveal a multistep pathway for closing of the substrate access channel .
  Biochemistry, 50, 693-703.  
20352666 C.Olano, C.Méndez, and J.A.Salas (2010).
Post-PKS tailoring steps in natural product-producing actinomycetes from the perspective of combinatorial biosynthesis.
  Nat Prod Rep, 27, 571-616.  
19902203 S.R.Park, A.R.Han, Y.H.Ban, Y.J.Yoo, E.J.Kim, and Y.J.Yoon (2010).
Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects.
  Appl Microbiol Biotechnol, 85, 1227-1239.  
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.  
20297780 Y.T.Lee, R.F.Wilson, I.Rupniewski, and D.B.Goodin (2010).
P450cam visits an open conformation in the absence of substrate.
  Biochemistry, 49, 3412-3419.
PDB codes: 3l61 3l62 3l63
  19342781 C.Hong, S.G.Bell, W.Yang, H.Wang, Y.Hao, X.Li, W.Zhou, M.Bartlam, and L.L.Wong (2009).
Purification, crystallization and preliminary X-ray analysis of cytochrome P450 219A1 from Novosphingobium aromaticivorans DSM 12444.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 65, 364-367.  
19625248 C.Savino, L.C.Montemiglio, G.Sciara, A.E.Miele, S.G.Kendrew, P.Jemth, S.Gianni, and B.Vallone (2009).
Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate.
  J Biol Chem, 284, 29170-29179.
PDB codes: 2jjn 2jjo 2wio
19555717 I.G.Denisov, D.J.Frank, and S.G.Sligar (2009).
Cooperative properties of cytochromes P450.
  Pharmacol Ther, 124, 151-167.  
18561189 J.G.McCoy, H.D.Johnson, S.Singh, C.A.Bingman, I.K.Lei, J.S.Thorson, and G.N.Phillips (2009).
Structural characterization of CalO2: a putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway.
  Proteins, 74, 50-60.
PDB code: 3buj
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
19124459 S.Li, H.Ouellet, D.H.Sherman, and L.M.Podust (2009).
Analysis of transient and catalytic desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces venezuelae.
  J Biol Chem, 284, 5723-5730.
PDB codes: 2vsj 2vz7 2vzm
19833867 S.Li, M.R.Chaulagain, A.R.Knauff, L.M.Podust, J.Montgomery, and D.H.Sherman (2009).
Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase.
  Proc Natl Acad Sci U S A, 106, 18463-18468.
PDB codes: 2whw 2wi9
  19342783 Y.Yasutake, Y.Fujii, W.K.Cheon, A.Arisawa, and T.Tamura (2009).
Crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase, a novel cytochrome P450 isolated from Pseudonocardia autotrophica.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 65, 372-375.  
18089574 H.Ouellet, L.M.Podust, and P.R.de Montellano (2008).
Mycobacterium tuberculosis CYP130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs.
  J Biol Chem, 283, 5069-5080.
PDB codes: 2uuq 2uvn
18838690 M.J.Cryle, and I.Schlichting (2008).
Structural insights from a P450 Carrier Protein complex reveal how specificity is achieved in the P450(BioI) ACP complex.
  Proc Natl Acad Sci U S A, 105, 15696-15701.
PDB codes: 3ejb 3ejd 3eje
18437375 P.Shrestha, T.J.Oh, K.Liou, and J.K.Sohng (2008).
Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin.
  Appl Microbiol Biotechnol, 79, 555-562.  
18245260 W.S.Jung, S.J.Jeong, S.R.Park, C.Y.Choi, B.C.Park, J.W.Park, and Y.J.Yoon (2008).
Enhanced heterologous production of desosaminyl macrolides and their hydroxylated derivatives by overexpression of the pikD regulatory gene in Streptomyces venezuelae.
  Appl Environ Microbiol, 74, 1972-1979.  
18804032 Y.Anzai, S.Li, M.R.Chaulagain, K.Kinoshita, F.Kato, J.Montgomery, and D.H.Sherman (2008).
Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
  Chem Biol, 15, 950-959.  
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.  
17915876 S.Li, L.M.Podust, and D.H.Sherman (2007).
Engineering and analysis of a self-sufficient biosynthetic cytochrome P450 PikC fused to the RhFRED reductase domain.
  J Am Chem Soc, 129, 12940-12941.  
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.

 

spacer
spacer