ENZYMOLOGY OF SQUALENE MONOOXYGENASE

Flavoprotein monooxygenases accomplish this oxygenation by forming a flavin hydroperoxide at the enzyme active site, which then transfers the terminal oxygen atom of the hydroperoxide (OH) to the substrate.  The remaining "hydroxyflavin" then reoxidizes with the release of water:

Squalene monooxygenase presumably utilizes this same mechanism, but differs from other known flavin monooxygenases in that the oxygen is inserted as an epoxide rather than as a hydroxyl group.  Indeed, this type of reaction is more typical of heme monooxygenases (the cytochromes P450) and some pterin-dependent hydroxylases. 

Squalene monooxygenase contains a loosely bound FAD flavin and obtains electrons from NADPH-cytochrome P450 reductase, rather than binding the nicotinamide cofactor NADPH directly.  This also distinguishes squalene monooxygenase from other flavin monooxygenases.  Because of the difficulty in purifying this microsomal enzyme, and its low abundance, even in liver, little is known about this enzyme.  My laboratory has cloned the human enzyme and undertaken the characterization of the enzyme after expression and purification from E. coli.  The abstract to our published work (described below) can be accessed here.   

As shown in the graph on the right, maximal monooxygenase activity is obtained at a reductase: monooxygenase ratio of approximately 1:3, indicating that electron transfer to the FAD group of squalene monooxygenase is not the rate-limiting step of the reaction. 

The Km of P450 reductase (14 nM) with squalene monooxygenase is low relative to that found for other P450 reductase redox partners, which are typically in the range of 10-500 nM.  Although this may reflect a strong interaction between P450 reductase and squalene monooxygenase, the 1:3 stoichiometry of the reaction probably biases this value.

Purified human squalene monooxygenase has a very poor rate of turnover for squalene, approximately 1 min^-1.  Similar rates have been obtained for the purified native and recombinant rat enzyme; these low rates may reflect the reconstitution conditions of the assay, including the use of Triton X-100 to replace uncharacterized cytosolic factor(s).  

The Km values for squalene (7.7 µM) and FAD (0.3 µM, lower graph) are similar to reported values for the human and rat enzymes.  The Km for FAD is unusually high, and reflects the ease with which the flavin dissociates from the enzyme.  

• Tai HH, Bloch K.  Squalene epoxidase of rat liver.  J Biol Chem 1972 Jun 25;247(12):3767-73

• Ono T, Bloch K.  Solubilization and partial characterization of rat liver squalene epoxidase.  J Biol Chem 1975 Feb 25;250(4):1571-9

• Ono T, Imai Y.  Squalene epoxidase from rat liver microsomes.  Methods Enzymol 1985;110:375-80

• Bai M, Prestwich GD.  Inhibition and activation of porcine squalene epoxidase.  Arch Biochem Biophys 1992 Mar;293(2):305-13 [abstract]

• Sakakibara J, Watanabe R, Kanai Y, Ono T.  Molecular cloning and expression of rat squalene epoxidase.  J Biol Chem 1995 Jan 6;270(1):17-20 [abstract]

• Nagumo A, Kamei T, Sakakibara J, Ono T.  Purification and characterization of recombinant squalene epoxidase.  J Lipid Res 1995 Jul;36(7):1489-97 [abstract]

• Lee HK, Denner-Ancona P, Sakakibara J, Ono T, Prestwich GD.  Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase.  Arch Biochem Biophys 2000 Sep 1;381(1):43-52 [abstract]

• Laden BP, Tang Y, Porter TD.  Cloning, Heterologous Expression, and Enzymological Characterization of Human Squalene Monooxygenase.  Arch Biochem Biophys 2000 Feb 15;374(2):381-388 [abstract]
  

For a tabulated database on the enzymology of squalene monooxygenase:

• BRENDA Enzyme Database entry for squalene monooxygenase

For other information on squalene monooxygenase:

• Biology of squalene monooxygenase

  • Discovery and Characterization

  • Regulation of Expression

  • Therapeutic and Natural Inhibitors
    
    

• The squalene monooxygenase family and related enzymes

  • Squalene Monooxygenase Sequences

  • Alignment of Squalene Monooxygenases

  • Other Flavoproteins Related to Squalene Monooxygenase

  • Comparison of Squalene Monooxygenase to p-Hydroxybenzoate Hydroxylase

  • The 3-Dimensional Structure of p-Hydroxybenzoate Hydroxylase
    
    

• Inhibition by tellurium and selenium compounds

  • Chemistry and Toxicity of Tellurium and Selenium

  • Inhibition of Squalene Monooxygenase by Tellurium and Selenium

  • Tellurium and Selenium Compounds React with Cysteines on Squalene Monooxygenase
     

   

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