title: The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate.
abstract: BACKGROUND
  Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes.
  
  
  RESULTS
  Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification.
  
  
  CONCLUSIONS
  Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides.
  
  
  CONCLUSIONS
  The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.
authors.affiliation: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Ebrecht
authors.affiliation: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Matías D
authors.initials: MD
authors.lastname: Asención Diez
authors.affiliation: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Claudia V
authors.initials: CV
authors.lastname: Piattoni
authors.affiliation: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Guerrero
authors.affiliation: Instituto de Agrobiotecnología del Litoral (UNL-CONICET), Facultad de Bioquímica y Ciencias Biológicas, Ciudad Universitaria, Paraje "El Pozo" CC 242, S3000ZAA Santa Fe, Argentina. Electronic address: iglesias@fbcb.unl.edu.ar.
authors.forename: Alberto A
authors.initials: AA
authors.lastname: Iglesias
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005695:Galactosephosphates / Q000378:metabolism*; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics; D005958:Glucosephosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism; D013997:Time Factors; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism*
chemical_list: D005695:Galactosephosphates; D005958:Glucosephosphates; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C029973:galactose-1-phosphate; D013879:Thioredoxins; C031590:glucose-1-phosphate; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D003545:Cysteine
keywords: Carbohydrate metabolism; Galactose-1-phosphate; Glucose-1-phosphate; Redox regulated; UDP-glucose pyrophosphorylase