Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica.
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.
Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan.
Ghulam
G
Jeelani
Afzal
A
Husain
Dan
D
Sato
Tomoyoshi
T
Soga
Makoto
M
Suematsu
Tomoyoshi
T
Nozaki
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D002413:Cations, Divalent; D017124:Conserved Sequence; D004748:Entamoeba histolytica / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011122:Polyphosphates / Q000737:chemistry; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species; D016415:Sequence Alignment; D013379:Substrate Specificity
D002413:Cations, Divalent; D011122:Polyphosphates; D015800:Protozoan Proteins; D017382:Reactive Oxygen Species; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D017853:Phosphotransferases (Alcohol Group Acceptor); D008274:Magnesium