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

Biochim Biophys Acta. 2015 Jan;1850(1):88-96. doi: 10.1016/j.bbagen.2014.10.002. Epub 2014 Oct 12.

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.

General significance: 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.

Keywords: Carbohydrate metabolism; Galactose-1-phosphate; Glucose-1-phosphate; Redox regulated; UDP-glucose pyrophosphorylase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biocatalysis
  • Cloning, Molecular
  • Cysteine / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Galactosephosphates / metabolism*
  • Giardia lamblia / enzymology*
  • Giardia lamblia / genetics
  • Glucosephosphates / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thioredoxins / metabolism
  • Time Factors
  • UTP-Glucose-1-Phosphate Uridylyltransferase / genetics
  • UTP-Glucose-1-Phosphate Uridylyltransferase / metabolism*

Substances

  • Galactosephosphates
  • Glucosephosphates
  • Protozoan Proteins
  • Recombinant Proteins
  • galactose-1-phosphate
  • Thioredoxins
  • glucose-1-phosphate
  • UTP-Glucose-1-Phosphate Uridylyltransferase
  • Cysteine