The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase.
Here, we have studied the role of a histidine residue with the lowest solvent accessibility among other histidine residues at the end of a short connecting structure (189AELH192) of the catalytic domain of the exo-inulinase through creation of H192A mutant. Site-directed mutagenesis method was applied to create the mutant enzyme. Molecular dynamics (MD) simulations, spectroscopic, calorimetric and kinetics analysis were used to study the structural and functional consequences of His192 substitution. Accordingly, the thermo-stabilities and catalytic performance were decreased upon H192A mutation. In silico and experimental approaches evidently confirm that His192 residue of exo-inulinase possesses structural and functional importance regardless of the lack of direct interaction with the substrate or involvement in the catalytic activity of exo-inulinase.
School of Biology, College of Science, University of Tehran, Tehran, Iran; Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Maryam Rezaei
MR
Arjomand
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. Electronic address: ahmadian@nigeb.ac.ir.
Gholamreza
G
Ahmadian
School of Biology, College of Science, University of Tehran, Tehran, Iran; Nano-Biomedicine Center of Excellence, Nanoscience and Nanotechnology Research Center, University of Tehran, Tehran, Iran. Electronic address: mhabibi@ut.ac.ir.
Mehran
M
Habibi-Rezaei
Department of Medicinal Chemistry & Drug Design and Development Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Malihe
M
Hassanzadeh
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Ali Asghar
AA
Karkhane
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran.
Ali Akbar
AA
Moosavi-Movahedi
Department of Medicinal Chemistry & Drug Design and Development Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Massoud
M
Amanlou
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D006639:Histidine; D006026:Glycoside Hydrolases; C023086:inulinase
Catalytic performance; Exo-inulinase; H192A mutant; Molecular dynamics; Thermo-stabilities