title: Molecular cloning and in silico studies of physiologically significant trehalase from Drosophila melanogaster.
abstract: Trehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55°C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure.
authors.affiliation: Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune 411008, India. Electronic address: e.shukla@ncl.res.in.
authors.forename: Ekta
authors.initials: E
authors.lastname: Shukla
authors.affiliation: Stress Biology Research Laboratory, Department of Zoology, S.P. Pune University, Pune 411007, India.
authors.forename: Leena
authors.initials: L
authors.lastname: Thorat
authors.affiliation: Cell and Molecular Biology Laboratory, Department of Biotechnology, S.P. Pune University, Pune 411007, India.
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhavnani
authors.affiliation: Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune 411008, India.
authors.forename: Ameya D
authors.initials: AD
authors.lastname: Bendre
authors.affiliation: Cell and Molecular Biology Laboratory, Department of Biotechnology, S.P. Pune University, Pune 411007, India.
authors.forename: J K
authors.initials: JK
authors.lastname: Pal
authors.affiliation: Stress Biology Research Laboratory, Department of Zoology, S.P. Pune University, Pune 411007, India.
authors.forename: B B
authors.initials: BB
authors.lastname: Nath
authors.affiliation: Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune 411008, India.
authors.forename: S M
authors.initials: SM
authors.lastname: Gaikwad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003198:Computer Simulation; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D062105:Molecular Docking Simulation; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014198:Trehalase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D014198:Trehalase
keywords: Glycoside hydrolase; Homology modeling; Molecular docking; Trehalase; Trehalose