The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate

• Published in: Biochimica et biophysica acta Vol. 1850; no. 1; pp. 88 - 96
• Main Authors: Ebrecht, Ana C., Asención Diez, Matías D., Piattoni, Claudia V., Guerrero, Sergio A., Iglesias, Alberto A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2015
• Subjects: Amino Acid Sequence; Biocatalysis; Carbohydrate metabolism; catalytic activity; Cloning, Molecular; cysteine; Cysteine - metabolism; DNA; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Galactose-1-phosphate; Galactosephosphates - metabolism; Giardia lamblia; Giardia lamblia - enzymology; Giardia lamblia - genetics; Glucose-1-phosphate; Glucosephosphates - metabolism; glycogen; heterologous gene expression; humans; hydrogen peroxide; Kinetics; metabolites; molecular cloning; Molecular Sequence Data; nitric oxide; oxidation; Oxidation-Reduction; parasites; pathogenicity; pathogens; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Recombinant Proteins - metabolism; Redox regulated; Sequence Homology, Amino Acid; Substrate Specificity; Thioredoxins - metabolism; Time Factors; transferases; UDP-glucose pyrophosphorylase; uridine triphosphate; UTP-Glucose-1-Phosphate Uridylyltransferase - genetics; UTP-Glucose-1-Phosphate Uridylyltransferase - metabolism; UDP-glucose pyrophosphorylase; Carbohydrate metabolism; Galactose-1-phosphate; Glucose-1-phosphate; Redox regulated
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2014.10.002

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. 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. 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. 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. •Giardia lamblia UDP-glucose pyrophosphorylase (recombinant) was characterized.•The enzyme activity was affected by redox compounds of physiological relevance.•The enzyme exhibited similar catalytic efficiency for glucose-1P and galactose-1P.•UDP-glucose pyrophosphorylase would be redox regulated.•The UDP-galactose produced by the enzyme would serve to the parasite metabolism.