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High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4.

Sevrioukova IF
Biochemistry 56 3058-3067 (2017)
Related entries: 5vc0, 5vcd, 5vce, 5vcg

Cited: 27 times
EuropePMC logo PMID: 28590129

Abstract

Human drug-metabolizing cytochrome P450 3A4 (CYP3A4) is a dynamic enzyme with a large and highly malleable active site that can fit structurally diverse compounds. Despite extensive investigations, structure-function relationships and conformational dynamics in CYP3A4 are not fully understood. This study was undertaken to engineer a well-expressed and functionally active cysteine-depleted CYP3A4 that can be used in biochemical and biophysical studies. cDNA codon optimization and screening mutagenesis were utilized to boost the level of bacterial expression of CYP3A4 and identify the least harmful substitutions for all six non-heme-ligating cysteines. The C58A/C64M/C98A/C239T/C377A/C468S (Cys-less) mutant was found to be expressed as highly as the optimized wild-type (opt-WT) CYP3A4. The high-resolution X-ray structures of opt-WT and Cys-less CYP3A4 revealed that gene optimization leads to a different folding in the Phe108 and Phe189 regions and promotes binding of the active site glycerol that interlocks Ser119 and Arg212, critical for ligand association, and the hydrophobic cluster adjacent to Phe108. Crowding and decreased flexibility of the active site, as well as structural alterations observed at the C64M, C239T, and C468S mutational sites, might be responsible for the distinct ligand binding behavior of opt-WT and Cys-less CYP3A4. Nonetheless, the Cys-less mutant could be used for structure-function investigations because it orients bromoergocryptine and ritonavir (a high-affinity substrate and a high-potency inhibitor, respectively) like the WT and has a higher activity toward 7-benzyloxy(4-trifluoromethyl)coumarin.

Articles - 5vcc mentioned but not cited (15)

  1. High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4. Sevrioukova IF. Biochemistry 56 3058-3067 (2017)
  2. Inhibition of Human CYP3A4 by Rationally Designed Ritonavir-Like Compounds: Impact and Interplay of the Side Group Functionalities. Samuels ER, Sevrioukova I. Mol Pharm 15 279-288 (2018)
  3. Interaction of Human Drug-Metabolizing CYP3A4 with Small Inhibitory Molecules. Sevrioukova I. Biochemistry 58 930-939 (2019)
  4. Structure-Activity Relationships of Rationally Designed Ritonavir Analogues: Impact of Side-Group Stereochemistry, Headgroup Spacing, and Backbone Composition on the Interaction with CYP3A4. Samuels ER, Sevrioukova I. Biochemistry 58 2077-2087 (2019)
  5. Rational Design of CYP3A4 Inhibitors: A One-Atom Linker Elongation in Ritonavir-Like Compounds Leads to a Marked Improvement in the Binding Strength. Samuels ER, Sevrioukova IF. Int J Mol Sci 22 E852 (2021)
  6. Testing the Pharmacokinetic Interactions of 24 Colonic Flavonoid Metabolites with Human Serum Albumin and Cytochrome P450 Enzymes. Mohos V, Fliszár-Nyúl E, Lemli B, Zsidó BZ, Hetényi C, Mladěnka P, Horký P, Pour M, Poór M. Biomolecules 10 E409 (2020)
  7. Photosensitive Ru(II) Complexes as Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4. Toupin N, Steinke SJ, Nadella S, Li A, Rohrabaugh TN, Samuels ER, Turro C, Sevrioukova IF, Kodanko JJ. J Am Chem Soc 143 9191-9205 (2021)
  8. Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6',7'-Dihydroxybergamottin. Sevrioukova IF. Int J Mol Sci 20 E4245 (2019)
  9. An increase in side-group hydrophobicity largely improves the potency of ritonavir-like inhibitors of CYP3A4. Samuels ER, Sevrioukova IF. Bioorg Med Chem 28 115349 (2020)
  10. Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4. Paquin A, Oufqir Y, Sevrioukova IF, Reyes-Moreno C, Bérubé G. Eur J Med Chem 220 113496 (2021)
  11. Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site. Sevrioukova IF. Int J Mol Sci 23 12591 (2022)
  12. Computational 3D Modeling-Based Identification of Inhibitors Targeting Cysteine Covalent Bond Catalysts for JAK3 and CYP3A4 Enzymes in the Treatment of Rheumatoid Arthritis. Faris A, Alnajjar R, Guo J, Al Mughram MH, Aouidate A, Asmari M, Elhallaoui M. Molecules 29 23 (2023)
  13. Interaction of CYP3A4 with caffeine: First insights into multiple substrate binding. Sevrioukova IF. J Biol Chem 299 105117 (2023)
  14. Network pharmacology and experimental validation to explore the potential mechanism of Sanjie Zhentong Capsule in endometriosis treatment. Guo R, Yi Z, Wang Y, Wang L. Front Endocrinol (Lausanne) 14 1110995 (2023)
  15. Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids. Tseilikman VE, Fedotova JO, Tseilikman OB, Novak J, Karpenko MN, Maistrenko VA, Lazuko SS, Belyeva LE, Kamel M, Buhler AV, Kovaleva EG. Int J Mol Sci 24 9333 (2023)


Reviews citing this publication (2)

  1. Targeting Metalloenzymes for Therapeutic Intervention. Chen AY, Adamek RN, Dick BL, Credille CV, Morrison CN, Cohen SM. Chem Rev 119 1323-1455 (2019)
  2. The relationships between cytochromes P450 and H2O2: Production, reaction, and inhibition. Albertolle ME, Peter Guengerich F. J Inorg Biochem 186 228-234 (2018)

Articles citing this publication (10)

  1. Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration. Albertolle ME, Phan TTN, Pozzi A, Guengerich FP. Mol Cell Proteomics 17 889-900 (2018)
  2. Structural Perspectives of the CYP3A Family and Their Small Molecule Modulators in Drug Metabolism. Wright WC, Chenge J, Chen T. Liver Res 3 132-142 (2019)
  3. Stereoselective Oxidation Kinetics of Deoxycholate in Recombinant and Microsomal CYP3A Enzymes: Deoxycholate 19-Hydroxylation Is an In Vitro Marker of CYP3A7 Activity. Chen YJ, Zhang J, Zhu PP, Tan XW, Lin QH, Wang WX, Yin SS, Gao LZ, Su MM, Liu CX, Xu L, Jia W, Sevrioukova IF, Lan K. Drug Metab Dispos 47 574-581 (2019)
  4. Conformational Response of N-Terminally Truncated Cytochrome P450 3A4 to Ligand Binding in Solution. Chuo SW, Liou SH, Wang LP, Britt RD, Poulos TL, Sevrioukova IF, Goodin DB. Biochemistry 58 3903-3910 (2019)
  5. Homotropic Cooperativity of Midazolam Metabolism by Cytochrome P450 3A4: Insight from Computational Studies. Li J, Chen Y, Tang Y, Li W, Tu Y. J Chem Inf Model 61 2418-2426 (2021)
  6. Mechanistic Insights into the Regio- and Stereoselectivities of Testosterone and Dihydrotestosterone Hydroxylation Catalyzed by CYP3A4 and CYP19A1. Li J, Tang Y, Li W, Tu Y. Chemistry 26 6214-6223 (2020)
  7. Steroid bioconjugation to a CYP3A4 allosteric site and its effect on substrate binding and coupling efficiency. Polic V, Sevrioukova IF, Auclair K. Arch Biochem Biophys 653 90-96 (2018)
  8. Interaction of CYP3A4 with Rationally Designed Ritonavir Analogues: Impact of Steric Constraints Imposed on the Heme-Ligating Group and the End-Pyridine Attachment. Samuels ER, Sevrioukova IF. Int J Mol Sci 23 7291 (2022)
  9. Investigating a new C2-symmetric testosterone dimer and its dihydrotestosterone analog: Synthesis, antiproliferative activity on prostate cancer cell lines and interaction with CYP3A4. Paquin A, Fortin L, Girouard J, Reyes-Moreno C, Sevrioukova IF, Bérubé G. Eur J Med Chem 250 115222 (2023)
  10. Nanodisc-embedded cytochrome P450 P3A4 binds diverse ligands by distributing conformational dynamics to its flexible elements. Paço L, Hackett JC, Atkins WM. J Inorg Biochem 244 112211 (2023)


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