Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants.
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.
Kobayashi
Y
Y
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson 85721, USA.
Fang
X
X
Szklarz
G D
GD
Halpert
J R
JR
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D001571:Benzoflavones / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003374:Coumarins / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000235:genetics* / Q000378:metabolism*; D003640:Dealkylation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D014633:Valine / Q000235:genetics
D001571:Benzoflavones; D003374:Coumarins; D010078:Oxazines; C014180:resorufin; D015660:7-Alkoxycoumarin O-Dealkylase; D019362:Cytochrome P-450 CYP2B1; D007930:Leucine; D014633:Valine; D000409:Alanine