Person:
MUTLU, ÖZAL

Profile Picture

Email Address

Birth Date

Research Projects

Organizational Units

OrgUnit Logo
Organizational Unit

Job Title

Last Name

MUTLU

First Name

ÖZAL

Name

Filters

2016 - 20193
Reset filters

Settings

Subject: BUPARVAQUONE ×

Search Results

Now showing 1 - 3 of 3
  • No Thumbnail Available
    PublicationMetadata only
    Biochemical and in silico Characterization of Recombinant L-Lactate Dehydrogenase of Theileria annulata
    (HUMANA PRESS INC, 2016) MUTLU, ÖZAL; Nural, Belma; Erdemir, Aysegul; Mutlu, Ozal; Yakarsonmez, Sinem; Danis, Ozkan; Topuzogullari, Murat; Turgut-Balik, Dilek
    Theileria annulata is a parasite that causes theileriosis in cattle. Reports about drug resistance made essential to develop new drug. LDH of Theileria schizonts is the vital enzyme for its anaerobic metabolism. TaLDH gene was first cloned into pGEM-T cloning vector with two introns in our previous study. Here we report cloning of TaLDH without introns into pLATE 31 vector in E. coli BL21(DE3). Protein was in an inactive form. Two mutations were fixed to express the active protein. Protein was purified by affinity chromatography and evaluated by SDS-PAGE and size exclusion chromatography. Optimum pH of enzyme was performed in pH 7.5, and enzyme was stabilized at 20-40 A degrees C. Enzyme kinetics of recombinant TaLDH were found to be in the direction of pyruvate to lactate K (m) 0.1324 and K (i) 4.295 mM, k (cat) , 44.55/s and k (cat) /K (m) , 3.3693 x 10(5)/M/s. 3D structure of TaLDH was predicted, and possible drug binding sites were determined by homology modelling.
  • No Thumbnail Available
    PublicationMetadata only
    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.
  • No Thumbnail Available
    PublicationMetadata only
    Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations
    (ELSEVIER SCIENCE INC, 2017) MUTLU, ÖZAL; Erdemir, Aysegul; Mutlu, Ozal
    Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. (C) 2017 Elsevier Inc. All rights reserved.