Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies

Chem Res Toxicol. 1995 Jun;8(4):574-9. doi: 10.1021/tx00046a011.


Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.

Publication types

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

MeSH terms

  • Androstenedione / metabolism
  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Base Sequence
  • Catalysis
  • Cell Membrane / enzymology
  • Chloramphenicol / metabolism
  • Chloramphenicol / pharmacology
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / biosynthesis*
  • Cytochrome P-450 Enzyme System / genetics*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics*
  • Liver / enzymology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Rats
  • Steroid Hydroxylases / antagonists & inhibitors
  • Steroid Hydroxylases / biosynthesis*
  • Steroid Hydroxylases / genetics*
  • Steroid Hydroxylases / metabolism
  • Substrate Specificity


  • Cytochrome P-450 Enzyme Inhibitors
  • Androstenedione
  • Chloramphenicol
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • steroid 16-beta-hydroxylase

Associated data

  • GENBANK/J00719
  • GENBANK/M11251