title: Periplasmic glucose-binding protein from Pseudomonas putida CSV86--identification of the glucose-binding pocket by homology-model-guided site-specific mutagenesis.
abstract: Glucose transport in Pseudomonas putida 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 [(14)C]-glucose uptake study revealed that glucose is transported by the high-affinity intracellular phosphorylative pathway. ppGBP was cloned, over-expressed in Escherichia coli 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 class II family of periplasmic binding proteins. The model showed highest structural similarity to GBP of Thermus thermophilus (ttGBP, rmsd 0.64 Å). 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 [(14)C]-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.
authors.affiliation: Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India.
authors.forename: Arnab
authors.initials: A
authors.lastname: Modak
authors.forename: Prasenjit
authors.initials: P
authors.lastname: Bhaumik
authors.forename: Prashant S
authors.initials: PS
authors.lastname: Phale
mesh_terms: D001692:Biological Transport; D020134:Catalytic Domain; D002942:Circular Dichroism; D005947:Glucose / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D019897:Periplasm / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000378:metabolism*; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000378:metabolism
chemical_list: D033902:Periplasmic Binding Proteins; D005947:Glucose
keywords: Pseudomonas; homology modeling; molecular docking; periplasmic binding protein; site-directed mutagenesis