In vitro cytochrome P450 46A1 (CYP46A1) activation by neuroactive compounds

• Published in: The Journal of biological chemistry Vol. 292; no. 31; pp. 12934 - 12946
• Main Authors: Mast, Natalia, Anderson, Kyle W., Johnson, Kevin M., Phan, Thanh T.N., Guengerich, F. Peter, Pikuleva, Irina A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.08.2017; American Society for Biochemistry and Molecular Biology
• Subjects: Acetylcholine - chemistry; Acetylcholine - metabolism; Alkynes; Allosteric Regulation - drug effects; Amino Acid Substitution; Anti-HIV Agents - chemistry; Anti-HIV Agents - metabolism; Anti-HIV Agents - pharmacology; Aspartic Acid - chemistry; Aspartic Acid - metabolism; Benzoxazines - chemistry; Benzoxazines - metabolism; Benzoxazines - pharmacology; Binding Sites; Biocatalysis - drug effects; central nervous system (CNS); cholesterol; Cholesterol 24-Hydroxylase - chemistry; Cholesterol 24-Hydroxylase - genetics; Cholesterol 24-Hydroxylase - metabolism; cholesterol metabolism; Cyclopropanes; cytochrome P450; Deuterium Exchange Measurement; Enzyme Activation - drug effects; enzyme catalysis; Enzymology; gamma-Aminobutyric Acid - chemistry; gamma-Aminobutyric Acid - metabolism; glutamate; Glutamic Acid - chemistry; Glutamic Acid - metabolism; Ligands; Models, Molecular; Molecular Docking Simulation; Mutagenesis, Site-Directed; Mutation; Nerve Tissue Proteins - agonists; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Conformation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; cholesterol; enzyme catalysis; cytochrome P450; central nervous system (CNS); cholesterol metabolism; glutamate
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.794909

Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase) is the enzyme responsible for the majority of cholesterol elimination from the brain. Previously, we found that the anti-HIV drug efavirenz (EFV) can pharmacologically activate CYP46A1 in mice. Herein, we investigated whether CYP46A1 could also be activated by endogenous compounds, including major neurotransmitters. In vitro experiments with purified recombinant CYP46A1 indicated that CYP46A1 is activated by l-glutamate (l-Glu), l-aspartate, γ-aminobutyric acid, and acetylcholine, with l-Glu eliciting the highest increase (3-fold) in CYP46A1-mediated cholesterol 24-hydroxylation. We also found that l-Glu and other activating neurotransmitters bind to the same site on the CYP46A1 surface, which differs from the EFV-binding site. The other principal differences between EFV and l-Glu in CYP46A1 activation include an apparent lack of l-Glu binding to the P450 active site and different pathways of signal transduction from the allosteric site to the active site. EFV and l-Glu similarly increased the CYP46A1 kcat, the rate of the “fast” phase of the enzyme reduction by the redox partner NADPH–cytochrome P450 oxidoreductase, and the amount of P450 reduced. Spectral titrations with cholesterol, in the presence of EFV or l-Glu, suggest that water displacement from the heme iron can be affected in activator-bound CYP46A1. Moreover, EFV and l-Glu synergistically activated CYP46A1. Collectively, our in vitro data, along with those from previous cell culture and in vivo studies by others, suggest that l-Glu-induced CYP46A1 activation is of physiological relevance.