title: Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach.
abstract: The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP(+) (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.
authors.affiliation: Molecular Biology and Proteomics Lab, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, Uttarakhand, India, rajbalasingh10@gmail.com.
authors.forename: Rajbala
authors.initials: R
authors.lastname: Yadav
authors.forename: Ramasare
authors.initials: R
authors.lastname: Prasad
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D006600:Hexosephosphates / Q000378:metabolism; D007700:Kinetics; D062105:Molecular Docking Simulation; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D006600:Hexosephosphates; D010940:Plant Proteins; D011994:Recombinant Proteins; C035752:sorbitol 6-phosphate; D013401:Sugar Alcohol Dehydrogenases; C022645:sorbitol-6-phosphate dehydrogenase