Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290–328 KJ/mol), except for the E308G variant that showed a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main Tm by 4–5 °C for all the proteins, exception made for E308G where no changes are observed. Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (Kd), enthalpy (ΔH) and entropy (ΔS). Kd values of 1.6 and 0.7 μM, ΔH of −13.9 Kcal/mol and −16.8 Kcal/mol, ΔS of −20.5 cal/mol/deg, and −28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the Tm results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the Tm stabilizing the structure of E158K (46.2 to 50.6 °C). Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme.

Ligand stabilization and effect on unfolding by polymorphism in human flavin-containing monooxygenase 3

Catucci G.;Sadeghi S. J.;Gilardi G.
2020

Abstract

Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290–328 KJ/mol), except for the E308G variant that showed a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main Tm by 4–5 °C for all the proteins, exception made for E308G where no changes are observed. Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (Kd), enthalpy (ΔH) and entropy (ΔS). Kd values of 1.6 and 0.7 μM, ΔH of −13.9 Kcal/mol and −16.8 Kcal/mol, ΔS of −20.5 cal/mol/deg, and −28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the Tm results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the Tm stabilizing the structure of E158K (46.2 to 50.6 °C). Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme.
162
1484
1493
Calorimetry; SNP; Unfolding
Catucci G.; Aramini D.; Sadeghi S.J.; Gilardi G.
File in questo prodotto:
File Dimensione Formato  
Catucci_pre-print.pdf

accesso aperto

Descrizione: Pdf preprint
Tipo di file: PREPRINT (PRIMA BOZZA)
Dimensione 1.16 MB
Formato Adobe PDF
1.16 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1769394
Citazioni
  • ???jsp.display-item.citation.pmc??? 3
  • Scopus 5
  • ???jsp.display-item.citation.isi??? 5
social impact