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Salemi, Zahra,Hosseinkhani, Saman,Ranjbar, Bijan,Nemat-Gorgani, Mohsen Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.5
Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. In the present study, an apo form of the enzyme was prepared by removal of zinc and a comparative study was performed on some characteristic features of the apo and native forms by far- and near-UV circular dichroism (CD), intrinsic fluorescent spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, fluorescence quenching by acrylamide, and Tm measurement. Results indicate that protein flexibility is enhanced and the hydrophobic sites become more exposed upon conversion to the apo form. Accordingly, the apo structure showed a greater affinity for interaction with hydrophobic adsorbents as compared with the native structure. As observed for the native enzyme, heat denaturation of the apo form promoted interaction with alkyl residues present on the adsorbents and, by cooling followed by addition of zinc, catalytically-active immobilized preparations were obtained.
cDNA Cloning, Expression and Homology Modeling of a Luciferase from the Firefly Lampyroidea maculata
Emamzadeh, Abdo Rahman,Hosseinkhani, Saman,Sadeghizadeh, Majid,Nikkhah, Maryam,Chaichi, Mohammad Javad,Mortazavi, Mojtaba Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.5
The cDNA of a firefly luciferase from lantern mRNA of Lampyroidea maculata has been cloned, sequenced and functionally expressed. The cDNA has an open reading frame of 1647 bp and codes for a 548-residue-long polypeptide. Noteworthy, sequence comparison as well as homology modeling showed the highest degree of similarity with H. unmunsana and L. mingrelica luciferases, suggesting a close phylogenetic relationship despite the geographical distance separation. The deduced amino acid sequence of the luciferase gene of firefly L. maculata showed 93% identity to H. unmunsana. Superposition of the three-dimensional model of L. maculata luciferase (generated by homology modeling) and three dimensional structure of Photinus pyralis luciferase revealed that the spatial arrangements of Luciferin and ATP-binding residues are very similar. Putative signature of AMP-binding domain among the various firefly species and Lampyroidea maculata was compared and a striking similarity was found. Different motifs and sites have been identified in Lampyroidea maculata by sequence analysis. Expression and purification of luciferase from Lampyroidea maculata was carried out using Ni-NTA Sepharose. Bioluminescence emission spectrum was similar to Photinus pyralis luciferase.
Alipour, Bagher Said,Hosseinkhani, Saman,Ardestani, Sussan K.,Moradi, Ali Korean Society of Photoscience 2009 Photochemical & photobiological sciences Vol.8 No.6
Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Amongst different beetle luciferases, those from phrixothrix rail-road worm with a unique additional residue (Arg353) emit red bioluminescence color naturally. Insertion of $Arg^{356}$ in Lampyris turkestanicus luciferase changed the emitted light to red with a bimodal bioluminescence spectrum. By insertion and substitution of positively-charged residues, different specific mutation (E354R/$Arg^{356}$, E354K/$Arg^{356}$, E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of $Arg^{356}$ produces a luciferase that emits red light with a single peak bioluminescence spectrum. The comparison of mutants with native luciferase shows that mutations of firefly luciferase resulted in structural and functional thermostability. Comparative study of native and mutant luciferase (E354R/$Arg^{356}$) by intrinsic and extrinsic fluorescence, CD spectropolarimetry, and homology modeling revealed mutation brought about an increase in content of secondary structure and globular compactness of L. turkestanicus luciferase. On the other hand, $pK_a$ of amino acids in the flexible loop decreased upon introducing of positive charges.
Shirazy, Najmeh Hadizadeh,Ranjbar, Bijan,Hosseinkhani, Saman,Khalifeh, Khosrow,Madvar, Ali Riahi,Naderi-Manesh, Hossein Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.4
Bacterial luciferase is a heterodimeric enzyme, which catalyzes the light emission reaction, utilizing reduced FMN (FMNH2), a long chain aliphatic aldehyde and $O_2$, to produce green-blue light. This enzyme can be readily classed as slow or fast decay based on their rate of luminescence decay in a single turnover. Mutation of Glu175 in $\alpha$ subunit to Gly converted slow decay Xenorhabdus Luminescence luciferase to fast decay one. The following studies revealed that changing the luciferase flexibility and lake of Glu-flavin interactions are responsible for the unusual kinetic properties of mutant enzyme. Optical and thermodynamics studies have caused a decrease in free energy and anisotropy of mutant enzyme. Moreover, the role of Glu175 in transition state of folding pathway by use of stopped-flow fluorescence technique has been studied which suggesting that Glu175 is not involved in transition state of folding and appears as surface residue of the nucleus or as a member of one of a few alternative folding nuclei. These results suggest that mutation of Glu175 to Gly extended the structure of Xenorhabdus Luminescence luciferase, locally.
( Khosrow Khalifeh ),( Bijan Ranjbar ),( Bagher Said Alipour ),( Saman Hosseinkhani ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.2
Thermodynamic stability and refolding kinetics of firefly luciferase and three representative mutants with depletion of negative charge on a flexible loop via substitution of Glu by Arg(ER mutant) or Lys (EK mutant) as well as insertion of another Arg in ER mutants (ERR mutant) was investigated. According to thermodynamic studies, structural stability of ERR and ER mutants are enhanced compared to WT protein, whereas, these mutants become prone to aggregation at higher temperatures. Accordingly, it was concluded that enhanced structural stability of mutants depends on more compactness of folded state, whereas aggregation at higher temperatures in mutants is due to weakening of intermolecular repulsive electrostatic interactions and increase of intermolecular hydrophobic interactions. Kinetic results indicate that early events of protein folding are accelerated in mutants. [BMB reports 2011; 44(2): 102-106]
Zonouzi, Roseata,Ashtiani, Saeid Kazemi,Hosseinkhani, Saman,Baharvand, Hossein Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.4
Embryonic stem cells (ESCs), representing a population of undifferentiated pluripotent cells with both self-renewal and multilineage differentiation characteristics, are capable of spontaneous differentiation into cardiomyocytes. The present study sought to define the kinetic characterization of lactate dehydrogenase (LDH) and creatine kinase (CK) of ESC- and neonatal-derived cardiomyocytes. Spontaneously differentiated cardiomyocytes from embryoid bodies (EBs) derived from mouse ESC line (Royan B1) and neonatal cardiomyocytes were dispersed in a buffer solution. Enzymes were extracted by sonication and centrifugation for kinetic evaluation of LDH and CK with spectrophotometric methods. While a comparison between the kinetic properties of the LDH and CK of both groups revealed not only different Michaelis constants and optimum temperatures for LDH but also different Michaelis constants and optimum pH for CK, the pH profile of LDH and optimum temperature of CK were similar. In defining some kinetic properties of cardiac metabolic enzymes of ESC-derived cardiomyocytes, our results are expected to further facilitate the use of ESCs as an experimental model.