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

Anna V. Glyakina, Nicolai I. Strizhov, Mikhail V. Karpov, Nikita V. Dovidchenko, Bakhyt Matkarimov, Ludmila V. Isaeva, Vera S. Efimova, Mikhail A. Rubtsov, Ludmila A. Novikova, Marina V. Donova, Oxana V. Galzitskaya

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)80-90
Number of pages11
JournalSteroids
DOIs
Publication statusPublished - Mar 1 2019

Fingerprint

Cholesterol Side-Chain Cleavage Enzyme
Catalyst activity
Cholesterol
Sterols
Enzymes
Conformations
Catalytic Domain
Amino Acids
Bioconversion
Pregnenolone
Mutagenesis
Mutation
Isoleucine
Enzyme activity
Amino Acid Substitution
Molecular Dynamics Simulation
Bioinformatics
Site-Directed Mutagenesis
Computational Biology
Leucine

Keywords

  • Catalytic activity
  • Cholesterol
  • MD simulations
  • Mutations
  • P450scc
  • β-Sitosterol

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry

Cite this

Glyakina, A. V., Strizhov, N. I., Karpov, M. V., Dovidchenko, N. V., Matkarimov, B., Isaeva, L. V., ... Galzitskaya, O. V. (2019). Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1). Steroids, 80-90. https://doi.org/10.1016/j.steroids.2019.01.002

Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1). / Glyakina, Anna V.; Strizhov, Nicolai I.; Karpov, Mikhail V.; Dovidchenko, Nikita V.; Matkarimov, Bakhyt; Isaeva, Ludmila V.; Efimova, Vera S.; Rubtsov, Mikhail A.; Novikova, Ludmila A.; Donova, Marina V.; Galzitskaya, Oxana V.

In: Steroids, 01.03.2019, p. 80-90.

Research output: Contribution to journalArticle

Glyakina, AV, Strizhov, NI, Karpov, MV, Dovidchenko, NV, Matkarimov, B, Isaeva, LV, Efimova, VS, Rubtsov, MA, Novikova, LA, Donova, MV & Galzitskaya, OV 2019, 'Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1)', Steroids, pp. 80-90. https://doi.org/10.1016/j.steroids.2019.01.002
Glyakina, Anna V. ; Strizhov, Nicolai I. ; Karpov, Mikhail V. ; Dovidchenko, Nikita V. ; Matkarimov, Bakhyt ; Isaeva, Ludmila V. ; Efimova, Vera S. ; Rubtsov, Mikhail A. ; Novikova, Ludmila A. ; Donova, Marina V. ; Galzitskaya, Oxana V. / Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1). In: Steroids. 2019 ; pp. 80-90.
@article{79bf4bdbf80e4a9a80f535d75d5fec11,
title = "Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1)",
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",
author = "Glyakina, {Anna V.} and Strizhov, {Nicolai I.} and Karpov, {Mikhail V.} and Dovidchenko, {Nikita V.} and Bakhyt Matkarimov and Isaeva, {Ludmila V.} and Efimova, {Vera S.} and Rubtsov, {Mikhail A.} and Novikova, {Ludmila A.} and Donova, {Marina V.} and Galzitskaya, {Oxana V.}",
year = "2019",
month = "3",
day = "1",
doi = "10.1016/j.steroids.2019.01.002",
language = "English",
pages = "80--90",
journal = "Steroids",
issn = "0039-128X",
publisher = "Elsevier",

}

TY - JOUR

T1 - Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1)

AU - Glyakina, Anna V.

AU - Strizhov, Nicolai I.

AU - Karpov, Mikhail V.

AU - Dovidchenko, Nikita V.

AU - Matkarimov, Bakhyt

AU - Isaeva, Ludmila V.

AU - Efimova, Vera S.

AU - Rubtsov, Mikhail A.

AU - Novikova, Ludmila A.

AU - Donova, Marina V.

AU - Galzitskaya, Oxana V.

PY - 2019/3/1

Y1 - 2019/3/1

N2 - 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.

AB - 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.

KW - Catalytic activity

KW - Cholesterol

KW - MD simulations

KW - Mutations

KW - P450scc

KW - β-Sitosterol

UR - http://www.scopus.com/inward/record.url?scp=85060290068&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85060290068&partnerID=8YFLogxK

U2 - 10.1016/j.steroids.2019.01.002

DO - 10.1016/j.steroids.2019.01.002

M3 - Article

C2 - 30641046

AN - SCOPUS:85060290068

SP - 80

EP - 90

JO - Steroids

JF - Steroids

SN - 0039-128X

ER -