title: Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures.
abstract: BACKGROUND
  The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy.
  
  
  METHODS
  The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii.
  
  
  RESULTS
  Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 μM and 38.3 ± 0.9 μM, respectively.
  
  
  CONCLUSIONS
  The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea. kuku1819@knu.ac.kr.
authors.forename: Youn-Kyoung
authors.initials: YK
authors.lastname: Goo
authors.affiliation: Research Center for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido, 001-0020, Japan. junya@czc.hokudai.ac.jp.
authors.forename: Junya
authors.initials: J
authors.lastname: Yamagishi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. akio.ueno@h-rise.jp.
authors.forename: Akio
authors.initials: A
authors.lastname: Ueno
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. alaaterkawi@obihiro.ac.jp.
authors.forename: Mohamad Alaa
authors.initials: MA
authors.lastname: Terkawi
authors.affiliation: Department of Public Health, Pharmacology and Toxicology, University of Nairobi, P.O. BOX 29053, 00625, Kangemi, Nairobi, Kenya. goluga@yahoo.co.uk.
authors.forename: Gabriel Oluga
authors.initials: GO
authors.lastname: Aboge
authors.affiliation: Department of Veterinary Medicine, College of Veterinary Medicine and Stem Cell Research Therapeutic Institute, Kyungpook National University, Daegu, 700-701, Republic of Korea. dmkwak@knu.ac.kr.
authors.forename: Dongmi
authors.initials: D
authors.lastname: Kwak
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea. ychong@knu.ac.kr.
authors.forename: Yeonchul
authors.initials: Y
authors.lastname: Hong
authors.affiliation: Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea. dichung@knu.ac.kr.
authors.forename: Dong-Il
authors.initials: DI
authors.lastname: Chung
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. makoto@obihiro.ac.jp.
authors.forename: Makoto
authors.initials: M
authors.lastname: Igarashi
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. nishikawa@obihiro.ac.jp.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nishikawa
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. gen@obihiro.ac.jp.
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003474:Curcumin / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000378:metabolism*; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000235:genetics* / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011765:Pyruvaldehyde / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014122:Toxoplasma / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D011994:Recombinant Proteins; D011765:Pyruvaldehyde; D007791:Lactoylglutathione Lyase; D005978:Glutathione; D003474:Curcumin