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 ∼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 ∼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 ∼32 kDa in promastigote extracts. By overexpressing the GLOII gene in Leishmania donovani using Leishmania expression vector pspαhygroα, 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.
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Research Article|
December 12 2005
Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs
Prasad K. Padmanabhan;
Prasad K. Padmanabhan
1School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
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Angana Mukherjee;
Angana Mukherjee
1School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
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Rentala Madhubala
Rentala Madhubala
1
1School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
June 14 2005
Revision Received:
August 18 2005
Accepted:
September 14 2005
Accepted Manuscript online:
September 14 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (1): 227–234.
Article history
Received:
June 14 2005
Revision Received:
August 18 2005
Accepted:
September 14 2005
Accepted Manuscript online:
September 14 2005
Citation
Prasad K. Padmanabhan, Angana Mukherjee, Rentala Madhubala; Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs. Biochem J 1 January 2006; 393 (1): 227–234. doi: https://doi.org/10.1042/BJ20050948
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