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MUTLU, ÖZAL

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MUTLU

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ÖZAL

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2012 - 20203
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    Functional analyses of dipeptide and pentapeptide insertions on Theileria annulata enolase by site-directed mutagenesis and in silico approaches
    (ELSEVIER SCIENCE BV, 2019) MUTLU, ÖZAL; Yakarsonmez, Sinem; Cengiz, Ebru Cayir; Mutlu, Ozal; Turgut-Balik, Dilek
    Theileria annulata enolase (TaENO) could be assessed as a druggable target for tropical theileriosis treatment. The parasite enzyme plays an important role in many cellular functions and carries some structural differences like dipeptide (262EK263) and pentapeptide ((103)EWGYC(107)) insertions from the host enzyme, Bos taurus enolase. In this study, the functional effects of these insertions on TaENO activity were analyzed by in vitro site-directed mutagenesis and in silico molecular docking analyses for the first time in the literature. In vitro results showed that, although the deletion of the pentapeptide insertion (TaENO Delta EWGYC) reduced the enzyme activity slightly, the removal of the dipeptide insertion (TaENO Delta EK) halted it. Also, molecular docking results revealed that the deletion of these insertions affected the substrate binding affinity of the mutant enzymes. The active site of TaENO Delta EK exhibited a small decrease of substrate binding affinity compared to the active site of TaENO Delta EWGYC relative to the wild type TaENO. Although we conclude that both regions could be evaluated as possible drug-binding sites to inhibit TaENO in further studies, these results indicate that the dipeptide insertion could be a more promising drug binding site than the pentapeptide insertion.
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    Discovery and evaluation of inhibitory activity and mechanism of arylcoumarin derivatives on Theileria annulata enolase by in vitro and molecular docking studies
    (SPRINGER, 2020) MUTLU, ÖZAL; Yakarsonmez, Sinem; Danis, Ozkan; Mutlu, Ozal; Topuzogullari, Murat; Sariyer, Emrah; Yuce-Dursun, Basak; Turgut-Balik, Dilek
    In this study, the inhibition potential of 3- and 4-arylcoumarin derivatives on Theileria annulata enolase (TaENO) was assessed for the first time in the literature. Firstly, protein stabilization analyses of TaENO were performed and it was found that the enzyme remains stable with the addition of 6 M ethylene glycol at + 4 degrees C. Inhibitor screening analyses were carried out using 25 coumarin derivatives on highly purified TaENO (> 95%), and four coumarin derivatives [4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (C8); 4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one (C9); 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one (C21); and 3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (C23)] showed the highest inhibitory effects with the IC50 values of 10.450, 13.170, 8.871 and 10.863 mu M, respectively. The kinetic results indicated that these compounds inhibited the enzyme by uncompetitive inhibition. In addition, the successful binding of the most potent inhibitor (C21) into TaENO was confirmed by using MALDI-TOF mass spectrophotometry. Molecular docking analyses have predicted that C8 and C21 coumarin derivatives which showed high inhibitory effects on TaENO were interacted with high affinity to the potential regions out of the active site. Taken together, these coumarin derivatives (C8, C9, C21 and C23) are first known potent, nonsubstrate, uncompetitive inhibitors of TaENO and these results will facilitate further in vitro and in vivo analysis toward structure-based drug design studies. [GRAPHICS] .
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    Kinetic Analysis of the Amino Terminal End of Active Site Loop of Lactate Deyhdrogenase from Plasmodium Vivax
    (GALENOS YAYINCILIK, 2012-12-22) MUTLU, ÖZAL; Mutlu, Ozal; Turgut-Balik, Dilek
    Objective: In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH's. Material and Methods: Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by Ni-NTA agarose matrix and kinetic properties were analysed. Results: Observing increase of K-m values of mutant enzymes showed that mutations in this place caused decreasing affinity of enzyme for its substrate. However k(cat) values were about the same throughout all mutant proteins. Conclusion: Sensitivity of the studied region emphasizes the significance of this site for drug design studies for both Plasmodium and some other Apicomplexans.