UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

Autor: Mabel Cristina Aleanzi, M.B. Bosco, Matías Machtey, Alberto A. Iglesias
Rok vydání: 2009
Předmět:
Models
Molecular
UDPGLUCOSE PYROPHOSPHORYLASE
Xanthomonas
UTP-Glucose-1-Phosphate Uridylyltransferase
Protein Conformation
Amino Acid Motifs
Genetic Vectors
Molecular Sequence Data
Biochemistry
Protein Structure
Secondary
Substrate Specificity
POLYSACCHARIDES
Protein structure
Escherichia coli
XANTHOMONAS
Amino Acid Sequence
Enzyme kinetics
Cloning
Molecular
Binding site
Conserved Sequence
chemistry.chemical_classification
Binding Sites
Molecular mass
biology
Otras Ciencias Químicas
Ciencias Químicas
Gene Amplification
General Medicine
biology.organism_classification
Recombinant Proteins
Protein Structure
Tertiary
Molecular Weight
Dissociation constant
Kinetics
OLIGOMERIC DISSOCIATION
Enzyme
chemistry
Genes
Bacterial
Transformation
Bacterial
Dimerization
CIENCIAS NATURALES Y EXACTAS
Plasmids
Protein Binding
UTP binding
Zdroj: Biochimie. 91:204-213
ISSN: 0300-9084
DOI: 10.1016/j.biochi.2008.09.001
Popis: The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60 kDa, exhibiting relatively high specific activity (∼80 Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15 μM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term. © 2008 Elsevier Masson SAS. All rights reserved. Fil: Bosco, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Machtey, Matías. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral; Argentina
Databáze: OpenAIRE
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