Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1

J Biol Chem. 2002 Oct 4;277(40):37582-9. doi: 10.1074/jbc.M204909200. Epub 2002 Jul 17.


Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Blotting, Western
  • Cholestanetriol 26-Monooxygenase
  • Cholesterol / metabolism*
  • Cloning, Molecular
  • Conserved Sequence
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Hydroxylation
  • Kinetics
  • Microsomes / enzymology*
  • Mitochondria / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Steroid Hydroxylases / chemistry
  • Steroid Hydroxylases / genetics*
  • Steroid Hydroxylases / metabolism*


  • Recombinant Proteins
  • Cholesterol
  • Steroid Hydroxylases
  • Cholestanetriol 26-Monooxygenase

Associated data

  • PDB/1MFX