Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs.
The glyoxalase system is a ubiquitous detoxification pathway that protects against cellular damage caused by highly reactive oxoaldehydes such as methylglyoxal which is mainly formed as a by-product of glycolysis. The gene encoding GLOII (glyoxalase II) has been cloned from Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. DNA sequence analysis revealed an ORF (open reading frame) of approximately 888 bp that encodes a putative 295-amino-acid protein with a calculated molecular mass of 32.5 kDa and a predicted pI of 6.0. The sequence identity between human GLOII and LdGLOII (L. donovani GLOII) is only 35%. The ORF is a single-copy gene on a 0.6-Mb chromosome. A approximately 38 kDa protein was obtained by heterologous expression of LdGLOII in Escherichia coli, and homogeneous enzyme was obtained after affinity purification. Recombinant L. donovani GLOII showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOII protein could detect a band of anticipated size approximately 32 kDa in promastigote extracts. By overexpressing the GLOII gene in Leishmania donovani using Leishmania expression vector pspalphahygroalpha, we detected elevated expression of GLOII RNA and protein. Overexpression of the GLOII gene will facilitate studies of gene function and its relevance as a chemotherapeutic target. This is the first report on the molecular characterization of glyoxalase II from Leishmania spp. The difference in the substrate specificity of the human and Leishmania donovani glyoxalase II enzyme could be exploited for structure-based drug design of selective inhibitors against the parasite.
Padmanabhan
Prasad K
PK
School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Mukherjee
Angana
A
Madhubala
Rentala
R
D000595:Amino Acid Sequence; D000818:Animals; D000977:Antiparasitic Agents / Q000494:pharmacology*; D017124:Conserved Sequence; D016503:Drug Delivery Systems; D007893:Leishmania donovani / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D000099301:Hydroxyacylglutathione Hydrolase
D000977:Antiparasitic Agents; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase