Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica

Ghulam Jeelani, Afzal Husain, Dan Sato, Tomoyoshi Soga, Makoto Suematsu, Tomoyoshi Nozaki

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6-2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.

Original languageEnglish
Pages (from-to)309-319
Number of pages11
JournalBiochimie
Volume95
Issue number2
DOIs
Publication statusPublished - 2013 Feb 1

Keywords

  • Entamoeba histolytica
  • NADH kinase
  • Nicotinamide (pyridine) nucleotide
  • Oxidative stress
  • Reactive oxygen species

ASJC Scopus subject areas

  • Biochemistry

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