PDBsum entry 1mxt

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Oxidoreductase PDB id
Protein chain
499 a.a. *
Waters ×738
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Atomic resolution structure of cholesterol oxidase (streptomyces sp. Sa-coo)
Structure: Cholesterol oxidase. Chain: a. Synonym: chod. Engineered: yes. Other_details: fad cofactor non-covalently bound to the enzyme
Source: Streptomyces sp.. Organism_taxid: 1931. Gene: choa. Expressed in: escherichia coli. Expression_system_taxid: 562.
0.95Å     R-factor:   0.110     R-free:   0.132
Authors: A.Vrielink,P.I.Lario
Key ref:
P.I.Lario et al. (2003). Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity. J Mol Biol, 326, 1635-1650. PubMed id: 12595270 DOI: 10.1016/S0022-2836(03)00054-8
03-Oct-02     Release date:   25-Feb-03    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P12676  (CHOD_STRS0) -  Cholesterol oxidase
546 a.a.
499 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class 2: E.C.  - Cholesterol oxidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Cholesterol + O2 = cholest-5-en-3-one + H2O2
Bound ligand (Het Group name = OXY)
corresponds exactly
= cholest-5-en-3-one
+ H(2)O(2)
      Cofactor: FAD
Bound ligand (Het Group name = FAE) corresponds exactly
   Enzyme class 3: E.C.  - Steroid Delta-isomerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A 3-oxo-Delta5-steroid = a 3-oxo-Delta4-steroid
= 3-oxo-Delta(4)-steroid
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!
  Cellular component     extracellular region   1 term 
  Biological process     oxidation-reduction process   4 terms 
  Biochemical function     oxidoreductase activity     6 terms  


DOI no: 10.1016/S0022-2836(03)00054-8 J Mol Biol 326:1635-1650 (2003)
PubMed id: 12595270  
Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity.
P.I.Lario, N.Sampson, A.Vrielink.
The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.
  Selected figure(s)  
Figure 1.
Figure 1. (a) The electron density map for the FAD cofactor and N485 modeled in two alternate conformations. The density was computed using the coefficients 2mFo 2 DFc and contoured at 1.0s (blue) and 4.0s (pink). Conformer B, shown in green, is 2.97 Å and 3.00 Å from both N3 and C4 of the FAD, respectively. (b) A superposition of the active site region of native cholesterol oxidase and the dehydroisoandosterone steroid complex from B. sterolicum (RCSB PDB accession codes 3COX and 1COY 10 ). The native structure is colored purple and the steroid complex structure is colored peach.
Figure 2.
Figure 2. Stereoscopic representations showing the tunnel linking the active site of the enzyme with the external environment of the protein. (a) A ribbon representation of the protein with the FAD binding domain shown in magenta and the substrate binding domain shown in blue. (b) The hydrophobic tunnel of the enzyme in the open conformation (corresponding to conformation B) and (c) the tunnel closed conformation (corresponding to conformation A). The tun- nel was visualized by constructing solvent-accessible surfaces with the program SPOCK 54 using a probe radius of 1.4 Å . The surface was colored according to residue type: aromatic and hydrophobic residues are colored green, acidic residues are colored red and basic residues are colored blue. The bound water molecules in the tunnel are represented as red spheres. The dehydroisoandosterone molecule, shown in green, has been modeled into the substrate-binding cavity.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 326, 1635-1650) copyright 2003.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19926290 H.X.Zhou, and J.A.McCammon (2010).
The gates of ion channels and enzymes.
  Trends Biochem Sci, 35, 179-185.  
19923719 A.Y.Lyubimov, L.Chen, N.S.Sampson, and A.Vrielink (2009).
A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase.
  Acta Crystallogr D Biol Crystallogr, 65, 1222-1231.
PDB codes: 3gyi 3gyj
19256550 I.Dreveny, A.S.Andryushkova, A.Glieder, K.Gruber, and C.Kratky (2009).
Substrate binding in the FAD-dependent hydroxynitrile lyase from almond provides insight into the mechanism of cyanohydrin formation and explains the absence of dehydrogenation activity.
  Biochemistry, 48, 3370-3377.
PDB codes: 3gdn 3gdp
19923715 I.S.Fernández, F.J.Ruíz-Dueñas, E.Santillana, P.Ferreira, M.J.Martínez, A.T.Martínez, and A.Romero (2009).
Novel structural features in the GMC family of oxidoreductases revealed by the crystal structure of fungal aryl-alcohol oxidase.
  Acta Crystallogr D Biol Crystallogr, 65, 1196-1205.
PDB code: 3fim
19843168 J.Kreit, and N.S.Sampson (2009).
Cholesterol oxidase: physiological functions.
  FEBS J, 276, 6844-6856.  
19574215 P.Ferreira, A.Hernandez-Ortega, B.Herguedas, A.T.Martínez, and M.Medina (2009).
Aryl-alcohol oxidase involved in lignin degradation: a mechanistic study based on steady and pre-steady state kinetics and primary and solvent isotope effects with two alcohol substrates.
  J Biol Chem, 284, 24840-24847.  
19541622 R.Baron, C.Riley, P.Chenprakhon, K.Thotsaporn, R.T.Winter, A.Alfieri, F.Forneris, W.J.van Berkel, P.Chaiyen, M.W.Fraaije, A.Mattevi, and J.A.McCammon (2009).
Multiple pathways guide oxygen diffusion into flavoenzyme active sites.
  Proc Natl Acad Sci U S A, 106, 10603-10608.  
18638483 D.S.Berkholz, H.R.Faber, S.N.Savvides, and P.A.Karplus (2008).
Catalytic cycle of human glutathione reductase near 1 A resolution.
  J Mol Biol, 382, 371-384.
PDB codes: 3djg 3djj 3dk4 3dk8 3dk9
18798008 F.M.Ho (2008).
Uncovering channels in photosystem II by computer modelling: current progress, future prospects, and lessons from analogous systems.
  Photosynth Res, 98, 503-522.  
18633481 J.F.Aparicio, and J.F.Martín (2008).
Microbial cholesterol oxidases: bioconversion enzymes or signal proteins?
  Mol Biosyst, 4, 804-809.  
18410129 L.Chen, A.Y.Lyubimov, L.Brammer, A.Vrielink, and N.S.Sampson (2008).
The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase.
  Biochemistry, 47, 5368-5377.
PDB code: 3cnj
18614534 L.Piubelli, M.Pedotti, G.Molla, S.Feindler-Boeckh, S.Ghisla, M.S.Pilone, and L.Pollegioni (2008).
  J Biol Chem, 283, 24738-24747.  
18029419 A.Y.Lyubimov, K.Heard, H.Tang, N.S.Sampson, and A.Vrielink (2007).
Distortion of flavin geometry is linked to ligand binding in cholesterol oxidase.
  Protein Sci, 16, 2647-2656.
PDB codes: 3b3r 3b6d
17675410 J.Saam, I.Ivanov, M.Walther, H.G.Holzhütter, and H.Kuhn (2007).
Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.
  Proc Natl Acad Sci U S A, 104, 13319-13324.  
17379143 M.V.Mendes, E.Recio, N.Antón, S.M.Guerra, J.Santos-Aberturas, J.F.Martín, and J.F.Aparicio (2007).
Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin.
  Chem Biol, 14, 279-290.  
17379137 N.M.Nesbitt, and N.S.Sampson (2007).
Antifungal tradecraft by cholesterol oxidase.
  Chem Biol, 14, 238-241.  
16604066 A.Y.Lyubimov, P.I.Lario, I.Moustafa, and A.Vrielink (2006).
Atomic resolution crystallography reveals how changes in pH shape the protein microenvironment.
  Nat Chem Biol, 2, 259-264.
PDB codes: 1n4u 1n4v 1n4w 2gew
17046020 I.M.Moustafa, S.Foster, A.Y.Lyubimov, and A.Vrielink (2006).
Crystal structure of LAAO from Calloselasma rhodostoma with an L-phenylalanine substrate: insights into structure and mechanism.
  J Mol Biol, 364, 991.
PDB code: 2iid
16999821 P.Ferreira, F.J.Ruiz-Dueñas, M.J.Martínez, W.J.van Berkel, and A.T.Martínez (2006).
Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme.
  FEBS J, 273, 4878-4888.  
16154992 C.H.Huang, W.L.Lai, M.H.Lee, C.J.Chen, A.Vasella, Y.C.Tsai, and S.H.Liaw (2005).
Crystal structure of glucooligosaccharide oxidase from Acremonium strictum: a novel flavinylation of 6-S-cysteinyl, 8alpha-N1-histidyl FAD.
  J Biol Chem, 280, 38831-38838.
PDB codes: 1zr6 2axr
15723552 E.C.Ralph, and P.F.Fitzpatrick (2005).
pH and kinetic isotope effects on sarcosine oxidation by N-methyltryptophan oxidase.
  Biochemistry, 44, 3074-3081.  
15661076 G.K.Kouassi, J.Irudayaraj, and G.McCarty (2005).
Examination of Cholesterol oxidase attachment to magnetic nanoparticles.
  J Nanobiotechnology, 3, 1.  
16127459 L.Puglielli, A.L.Friedlich, K.D.Setchell, S.Nagano, C.Opazo, R.A.Cherny, K.J.Barnham, J.D.Wade, S.Melov, D.M.Kovacs, and A.I.Bush (2005).
Alzheimer disease beta-amyloid activity mimics cholesterol oxidase.
  J Clin Invest, 115, 2556-2563.  
16332885 M.H.Lee, W.L.Lai, S.F.Lin, C.S.Hsu, S.H.Liaw, and Y.C.Tsai (2005).
Structural characterization of glucooligosaccharide oxidase from Acremonium strictum.
  Appl Environ Microbiol, 71, 8881-8887.  
15861372 S.Liu, U.Wollenberger, J.Halámek, E.Leupold, W.Stöcklein, A.Warsinke, and F.W.Scheller (2005).
Affinity interactions between phenylboronic acid-carrying self-assembled monolayers and flavin adenine dinucleotide or horseradish peroxidase.
  Chemistry, 11, 4239-4246.  
15163408 E.Gross, D.B.Kastner, C.A.Kaiser, and D.Fass (2004).
Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell.
  Cell, 117, 601-610.
PDB codes: 1rp4 1rq1
15128738 H.Khan, T.Barna, R.J.Harris, N.C.Bruce, I.Barsukov, A.W.Munro, P.C.Moody, and N.S.Scrutton (2004).
Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation.
  J Biol Chem, 279, 30563-30572.
PDB codes: 1vyp 1vyr 1vys
15194705 J.D.Phillips, F.G.Whitby, C.A.Warby, P.Labbe, C.Yang, J.W.Pflugrath, J.D.Ferrara, H.Robinson, J.P.Kushner, and C.P.Hill (2004).
Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae.
  J Biol Chem, 279, 38960-38968.
PDB codes: 1tk1 1tkl 1tlb
14739317 M.M.Teeter (2004).
Myoglobin cavities provide interior ligand pathway.
  Protein Sci, 13, 313-318.  
14675549 A.Vrielink, and N.Sampson (2003).
Sub-Angstrom resolution enzyme X-ray structures: is seeing believing?
  Curr Opin Struct Biol, 13, 709-715.  
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.