Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants

Biochemistry. 1998 May 12;37(19):6679-88. doi: 10.1021/bi9731450.


A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7. The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds. In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin. The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation. The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed. HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin. Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 7-Alkoxycoumarin O-Dealkylase / metabolism
  • Alanine / genetics
  • Animals
  • Benzoflavones / metabolism
  • Binding Sites / genetics
  • Coumarins / metabolism
  • Cytochrome P-450 CYP2B1 / genetics*
  • Cytochrome P-450 CYP2B1 / metabolism*
  • Dealkylation
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Leucine / genetics
  • Models, Molecular
  • Mutagenesis, Site-Directed*
  • Oxazines / metabolism
  • Oxidation-Reduction
  • Rats
  • Valine / genetics


  • Benzoflavones
  • Coumarins
  • Oxazines
  • resorufin
  • 7-Alkoxycoumarin O-Dealkylase
  • Cytochrome P-450 CYP2B1
  • Leucine
  • Valine
  • Alanine