Interconversion of the androstenedione hydroxylase specificities of cytochromes P450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues

Arch Biochem Biophys. 1996 Nov 1;335(1):152-60. doi: 10.1006/abbi.1996.0493.


Based on recent studies of single reciprocal mutants of cytochrome P450 2B4 and the highly related P450 2B5 at positions 114, 294, 363, and 367 [G. D. Szklarz, Y. Q. He, K. M. Kedzie, J. R. Halpert, and V. L. Burnett (1996) Arch. Biochem. Biophys. 327,308-318], a number of multiple mutants were constructed, expressed in Escherichia coli, and assayed with androstenedione, progesterone, and benzyloxyresorufin. Simultaneous substitutions of Ile-114, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 with Phe, Thr, Val, and Ala, respectively from 2B5, resulted in a marked increase in androstenedione 15alpha- and 16alpha-hydroxylation compared with the wild-type enzyme and yielded a metabolite profile indistinguishable from that of cytochrome P450 2B5. Likewise, the reciprocal P450 2B5 quadruple mutant exhibited the specificity for 16beta-hydroxylation characteristic of the 2B4 wild type. The two reciprocal quadruple mutants of P450 2B4 and 2B5 also displayed benzyloxyresorufin dealkylase activities similar to those of the wild-type P450 2B5 and 2B4, respectively. However, the progesterone metabolite profile of P450 2B5 was not identical to that of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue. Therefore, the 17beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.

Publication types

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

MeSH terms

  • Androstenedione / metabolism
  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Base Sequence
  • Binding Sites
  • Cloning, Molecular
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / metabolism*
  • Cytochrome P450 Family 2
  • DNA Primers
  • Escherichia coli
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oxazines / metabolism
  • Point Mutation
  • Polymerase Chain Reaction
  • Progesterone / metabolism
  • Protein Conformation
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Steroid 16-alpha-Hydroxylase
  • Steroid Hydroxylases / chemistry
  • Steroid Hydroxylases / metabolism*
  • Substrate Specificity


  • DNA Primers
  • Oxazines
  • Recombinant Proteins
  • Androstenedione
  • Progesterone
  • benzyloxyresorufin
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P450 Family 2
  • Steroid 16-alpha-Hydroxylase
  • cytochrome P-450 CYP2B5 (rabbit)
  • steroid 15-alpha-hydroxylase