Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4

Arch Biochem Biophys. 1996 Mar 15;327(2):308-18. doi: 10.1006/abbi.1996.0127.


The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation. Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile-114--> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Codon
  • Computer Simulation
  • Cytochrome P-450 Enzyme System / biosynthesis
  • 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
  • Point Mutation
  • Polymerase Chain Reaction
  • Protein Conformation*
  • Protein Multimerization
  • Rabbits
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Steroid Hydroxylases / biosynthesis
  • Steroid Hydroxylases / chemistry*
  • Steroid Hydroxylases / metabolism*
  • Transfection


  • Codon
  • DNA Primers
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P450 Family 2
  • cytochrome P-450 CYP2B5 (rabbit)
  • steroid 15-alpha-hydroxylase