Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
Permanent URI for this collectionhttps://hdl.handle.net/11147/7148
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Article Citation - WoS: 2Citation - Scopus: 2Small Angle X-Ray Scattering Analysis of Thermophilic Cytochrome P450 Cyp119 and the Effects of the N-Terminal Histidine Tag(Elsevier B.V., 2024) Doğru,E.K.; Sakallı,T.; Liu,G.; Sayers,Z.; Surmeli,N.B.Combining size exclusion chromatography-small angle X-ray scattering (SEC-SAXS) and molecular dynamics (MD) analysis is a promising approach to investigate protein behavior in solution, particularly for understanding conformational changes due to substrate binding in cytochrome P450s (CYPs). This study investigates conformational changes in CYP119, a thermophilic CYP from Sulfolobus acidocaldarius that exhibits structural flexibility similar to mammalian CYPs. Although the crystal structure of ligand-free (open state) and ligand-bound (closed state) forms of CYP119 is known, the overall structure of the enzyme in solution has not been explored until now. It was found that theoretical scattering profiles from the crystal structures of CYP119 did not align with the SAXS data, but conformers from MD simulations, particularly starting from the open state (46 % of all frames), agreed well. Interestingly, a small percentage of closed-state conformers also fit the data (9 %), suggesting ligand-free CYP119 samples ligand-bound conformations. Ab initio SAXS models for N-His tagged CYP119 revealed a tail-like unfolded structure impacting protein flexibility, which was confirmed by in silico modeling. SEC-SAXS analysis of N-His CYP119 indicated pentameric structures in addition to monomers in solution, affecting the stability and activity of the enzyme. This study adds insights into the conformational dynamics of CYP119 in solution. © 2024 Elsevier B.V.Article Citation - WoS: 4Citation - Scopus: 6Rational Design of Thermophilic Cyp119 for Progesterone Hydroxylation by in Silico Mutagenesis and Docking Screening(Elsevier, 2023) Kestevur Doğru, Ekin; Güralp, Gülce; Uyar, Arzu; Sürmeli, Nur BaşakSteroid-based chemicals can affect the metabolism, immune functions, and development of sexual characteristics. Because of these effects, steroid derivatives are widely used in the pharmaceutical industry. Progesterone is a steroid-based hormone that mainly controls the ovulation period of women but is also a precursor molecule for the synthesis of important hormones like testosterone and cortisone. Cytochrome P450 (CYP) enzymes are important for the production of hydroxyprogesterones in the industry since they can catalyze regio- and enantioselective hydroxylation reactions. Although human CYP enzymes can catalyze hydroxyprogesterone synthesis with high selectivity, these enzymes are membrane bound, which limits their application for industrial production. CYP119 is a soluble and thermophilic enzyme from the archaea Sulfolobus acidocaldarius. Even though the native substrate of the enzyme is not known, CYP119 can catalyze styrene epoxidation, lauric acid hydroxylation, and Amplex®Red peroxidation. In this work, an in silico mutagenesis approach was used to design CYP119 mutants with high progesterone affinity. Energy scores of progesterone docking simulations were used for the design and elimination of single, double, and triple mutants of CYP119. Among designed 674 mutants, five of them match the criteria for progesterone hydroxylation. The most common mutation of these five mutants, L69G mutant was analyzed using independent molecular dynamics (MD) simulations in comparison with the wild-type (WT) enzyme. L69G CYP119, was expressed and isolated from Escherichia coli; it showed 800-fold higher affinity for progesterone compared to WT CYP119. L69G CYP119 also catalyzed progesterone hydroxylation. The novel designed enzyme L69G CYP119 is a potential versatile biocatalyst for progesterone hydroxylation that is expected to be stable under industrial production conditions.