Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1)

Steroids. 2019 Mar;143:80-90. doi: 10.1016/j.steroids.2019.01.002. Epub 2019 Jan 11.

Abstract

Cytochrome P450scc (CYP11A1) is a mammalian mitochondrial enzyme which catalyzes cholesterol side chain cleavage to form pregnenolone. Along with cholesterol, some other steroids including sterols with a branched side chain like β-sitosterol are the substrates for the enzyme, but the activity towards β-sitosterol is rather low. Modification of the catalytic site conformation could provide more effective β-sitosterol bioconversion by the enzyme. This study was aimed to find out the amino acid residues substitution of which could modify the conformation of the active site providing possible higher enzyme activity towards β-sitosterol. After structural and bioinformatics analysis three amino acid residues I351, L355, I461 were chosen. Molecular dynamics simulations of P450scc evidenced the stability of the wild type, double (I351A/L355A) and triple (I351A/L355A/I461A) mutants. Mutant variants of cDNA encoding P450scc with the single, double and triple mutations were obtained by site-directed mutagenesis. However, the experimental data indicate that the introduced single mutations Ile351A, Leu355A and Ile461A dramatically decrease the target catalytic activity of CYP11A1, and no activity was observed for double and triple mutants obtained. Therefore, isoleucine residues 351 and 461, and leucine residue 355 are important for the cytochrome P450scc functioning towards sterols both with unbranched (cholesterol) and branched (sitosterol) side chains.

Keywords: Catalytic activity; Cholesterol; MD simulations; Mutations; P450scc; β-Sitosterol.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biocatalysis*
  • Cattle
  • Cholesterol / metabolism
  • Cholesterol Side-Chain Cleavage Enzyme / chemistry*
  • Cholesterol Side-Chain Cleavage Enzyme / genetics
  • Cholesterol Side-Chain Cleavage Enzyme / metabolism*
  • Molecular Dynamics Simulation
  • Mutagenesis
  • Protein Conformation

Substances

  • Cholesterol
  • Cholesterol Side-Chain Cleavage Enzyme