PDBsum entry 1ijh

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protein ligands links
Oxidoreductase PDB id
Protein chain
498 a.a. *
Waters ×607
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Cholesterol oxidase from streptomyces asn485leu mutant
Structure: Cholesterol oxidase. Chain: a. Engineered: yes. Mutation: 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.
1.53Å     R-factor:   0.166     R-free:   0.213
Authors: A.Vrielink,P.I.Lario
Key ref:
Y.Yin et al. (2001). The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase. Biochemistry, 40, 13779-13787. PubMed id: 11705367 DOI: 10.1021/bi010843i
26-Apr-01     Release date:   28-Dec-01    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P12676  (CHOD_STRS0) -  Cholesterol oxidase
546 a.a.
498 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.  - Cholesterol oxidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Cholesterol + O2 = cholest-5-en-3-one + H2O2
+ O(2)
= cholest-5-en-3-one
+ H(2)O(2)
      Cofactor: FAD
Bound ligand (Het Group name = FAD) 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: 10.1021/bi010843i Biochemistry 40:13779-13787 (2001)
PubMed id: 11705367  
The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase.
Y.Yin, N.S.Sampson, A.Vrielink, P.I.Lario.
Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.

Literature references that cite this PDB file's key reference

  PubMed id Reference
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
19045881 H.Takahashi, H.Ohno, R.Kishi, M.Nakano, and N.Matubayasi (2008).
Computation of the free energy change associated with one-electron reduction of coenzyme immersed in water: a novel approach within the framework of the quantum mechanical/molecular mechanical method combined with the theory of energy representation.
  J Chem Phys, 129, 205103.  
18001769 J.E.Lee, E.Bae, C.A.Bingman, G.N.Phillips, and R.T.Raines (2008).
Structural basis for catalysis by onconase.
  J Mol Biol, 375, 165-177.
PDB codes: 2gmk 2i5s
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
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
17379137 N.M.Nesbitt, and N.S.Sampson (2007).
Antifungal tradecraft by cholesterol oxidase.
  Chem Biol, 14, 238-241.  
17050691 J.Zhang, F.E.Frerman, and J.J.Kim (2006).
Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool.
  Proc Natl Acad Sci U S A, 103, 16212-16217.
PDB codes: 2gmh 2gmj
12547421 V.Anantharaman, L.Aravind, and E.V.Koonin (2003).
Emergence of diverse biochemical activities in evolutionarily conserved structural scaffolds of proteins.
  Curr Opin Chem Biol, 7, 12-20.  
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.