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Periplasmic glucose-binding protein from Pseudomonas putida CSV86-identification of the glucose-binding pocket by homology-model-guided site-specific mutagenesis

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Title Periplasmic glucose-binding protein from Pseudomonas putida CSV86-identification of the glucose-binding pocket by homology-model-guided site-specific mutagenesis
 
Creator MODAK, A
BHAUMIK, P
PHALE, PS
 
Subject homology modeling
molecular docking
periplasmic binding protein
Pseudomonas
site-directed mutagenesis
CARBON CATABOLITE REPRESSION
CRYSTAL-STRUCTURES
PREFERENTIAL UTILIZATION
SECONDARY-STRUCTURE
AROMATIC-COMPOUNDS
ESCHERICHIA-COLI
TRAP TRANSPORTER
AERUGINOSA
RECEPTOR
CSV86
 
Description Glucose transport in Pseudomonasputida CSV86 is mediated via a periplasmic glucose-binding protein (GBP)-dependent putative glucose ABC transporter. Here we describe a homology model and functional characterization of GBP from CSV86 (ppGBP). A whole-cell [C-14]-glucose uptake study revealed that glucose is transported by the high-affinity intracellular phosphorylative pathway. ppGBP was cloned, over-expressed in Escherichiacoli and purified to apparent homogeneity. The purified ppGBPs from both E.coli and CSV86 were found to be specific for glucose. A homology model of ppGBP was constructed that resembles the classII family of periplasmic binding proteins. The model showed highest structural similarity to GBP of Thermusthermophilus (ttGBP, rmsd 0.64 angstrom). Structural analysis and molecular docking studies predicted W35, W36, E41, K92, K339 and H379 of ppGBP as putative glucose-binding residues. Alanine substitution of these residues resulted in significantly reduced [C-14]-glucose binding activity. Analysis of the operonic arrangement and structural comparative studies suggested that ppGBP and ttGBP probably originated from a common ancestor. Structural adaptations that inhibit binding of di- or trisaccharides at the glucose-binding pocket of ppGBP were also identified.
 
Publisher WILEY-BLACKWELL
 
Date 2014-12-28T14:55:53Z
2014-12-28T14:55:53Z
2014
 
Type Article
 
Identifier FEBS JOURNAL, 281(1)365-375
1742-464X
1742-4658
http://dx.doi.org/10.1111/febs.12607
http://dspace.library.iitb.ac.in/jspui/handle/100/16808
 
Language English