Structural determinants of progesterone hydroxylation by cytochrome P450 2B5: the role of nonsubstrate recognition site residues

Arch Biochem Biophys. 1998 Feb 15;350(2):333-9. doi: 10.1006/abbi.1997.0516.


The highly related rabbit cytochromes P450 2B4 and 2B5 differ in only 12 amino acid positions, but only 2B5 has activity toward progesterone. Previously, simultaneous site-directed mutagenesis of four key substrate recognition site (SRS) residues (114, 294, 363, and 367) was shown to result in interconversion of the androstenedione hydroxylase specificities of cytochrome P450 2B4 and 2B5. However, the progesterone metabolite profiles of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue were not identical to that of P450 2B5. Therefore, single mutants of P450 2B5 at the remaining seven positions were constructed, expressed in Escherichia coli, and studied with progesterone as the substrate. The single mutants at positions 120 and 221, which are outside any known SRS, exhibited a significant alteration in progesterone hydroxylation. Based on these results, Ile-114, Arg-120, Ser-221, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 were replaced simultaneously with Phe, His, Pro, Thr, Val, and Ala, respectively, from 2B5. This yielded a mutant with a very similar progesterone metabolite profile to that of 2B5, although the total activity was lower. An additional substitution at residue 370 produced a multiple mutant P450 2B4 I114F-R120H-S221P-S294T-I363V-V367A- T370M with very similar or identical substrate specificity, regio- and stereospecificity and kinetic properties to that of P450 2B5 wild type.

Publication types

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

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Cytochrome P450 Family 2
  • Escherichia coli / genetics
  • Hydroxylation
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed / genetics
  • Progesterone / metabolism*
  • Rabbits
  • Recombinant Proteins / chemistry
  • Steroid Hydroxylases / chemistry*
  • Steroid Hydroxylases / genetics
  • Steroid Hydroxylases / metabolism


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