Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
Permanent URI for this collectionhttps://hdl.handle.net/11147/7148
Browse
6 results
Search Results
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.Article Citation - WoS: 6Citation - Scopus: 6Functional Characterization of a Novel Cyp119 Variant To Explore Its Biocatalytic Potential(Wiley, 2021) Sakallı, Tuğçe; Sürmeli, Nur BaşakBiocatalysts are increasingly applied in the pharmaceutical and chemical industry. Cytochrome P450 enzymes (P450s) are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. P450s catalyze reactions using nicotinamide adenine dinucleotide phosphate (NAD(P)H) cofactor and electron transfer proteins. Alternatively, P450s can utilize hydrogen peroxide (H2O2) as an oxidant, but this pathway is inefficient. P450s that show higher efficiency with peroxides are sought after in industrial applications. P450s from thermophilic organisms have more potential applications as they are stable toward high temperature, high and low pH, and organic solvents. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius. In our previous study, a novel T213R/T214I (double mutant [DM]) variant of CYP119 was obtained by screening a mutant library for higher peroxidation activity utilizing H2O2. Here, we characterized the substrate scope; stability toward peroxides; and temperature and organic solvent tolerance of DM CYP119 to identify its potential as an industrial biocatalyst. DM CYP119 displayed higher stability than wild-type (WT) CYP119 toward organic peroxides. It shows higher peroxidation activity for non-natural substrates and higher affinity for progesterone and other bioactive potential substrates compared to WT CYP119. DM CYP119 emerges as a new biocatalyst with a wide range of potential applications in the pharmaceutical and chemical industry.Article Citation - WoS: 2Citation - Scopus: 2Cloning, Expression, and Characterization of a Novel Sericin-Like Protein(Wiley, 2022) Bostan, Fatmanur; Sürmeli, Nur BaşakSilk consists of two proteins called fibroin and sericin. While fibroin is used in the textile industry and has various biomaterial applications, sericin has been considered as waste material until recently. Sericin is a multicomponent protein and it has important properties such as biocompatibility, biodegradability, cryoprotectivity, and antioxidant. Sericin from silkworm cocoons can be obtained by chemical, enzymatic, and heat treatment methods. However, sericin obtained with these treatment methods is not of consistent and high quality. Moreover, the exposure of sericin to harsh conditions during extraction leads to inconsistencies in the composition and structure of the sericin obtained. The inconsistencies in sericin structure and composition decrease application of sericin as a biomaterial. Here, we produce a sericin-like protein (Ser4mer) with native sequence of sericin encoding four repeats of the conserved 38 amino acid motif recombinantly in Escherichia coli and characterize its structural properties. Ser4mer protein shows similar structure to native sericin and higher solubility than previously obtained recombinant sericin-like proteins. Recombinant production of a soluble sericin-like protein will significantly expand its applications as a biomaterial. In addition, recombinant production of silk proteins will allow us to understand sequence-structure relationships in these proteins.Article Citation - WoS: 9Citation - Scopus: 9Development of an Improved Amplex Red Peroxidation Activity Assay for Screening Cytochrome P450 Variants and Identification of a Novel Mutant of the Thermophilic Cyp119(Springer, 2020) Başlar, M. Semih; Sakallı, Tuğçe; Güralp, Gülce; Kestevur Doğru, Ekin; Haklı, Emre; Sürmeli, Nur BaşakBiocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon-hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H2O2) as an oxygen and electron donor, but the reaction with H(2)O(2)is inefficient. The development of P450s that can use H(2)O(2)will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H2O2-dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 fromSulfolobus acidocaldarius, with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higherk(cat)for Amplex Red peroxidation and twofold higherk(cat)for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H2O2. While theK(m)for H(2)O(2)and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity. [GRAPHICS] .Article Citation - WoS: 33Citation - Scopus: 34Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 Cyp119(Hindawi Publishing Corporation, 2019) Aslantaş, Yaprak; Sürmeli, Nur BaşakBiocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of "unactivated" carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.Article Citation - WoS: 3Citation - Scopus: 5A Novel Thermophilic Hemoprotein Scaffold for Rational Design of Biocatalysts(Springer Verlag, 2018) Efua Aggrey Fynn, Joana; Sürmeli, Nur BaşakHemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze H2O2 decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by H2O2. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with H2O2, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design. [GRAPHICS] .