A glutathione-specific aldose reductase of Leishmania donovani and its potential implications for methylglyoxal detoxification pathway.
Methylglyoxal is mainly catabolized by two major enzymatic pathways. The first is the ubiquitous detoxification pathway, the glyoxalase pathway. In addition to the glyoxalase pathway, aldose reductase pathway also plays a crucial role in lowering the levels of methylglyoxal. The gene encoding aldose reductase (ALR) has been cloned from Leishmania donovani, a protozoan parasite causing visceral leishmaniasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 855 bp encoding a putative protein of 284 amino acids with a calculated molecular mass of 31.7 kDa and a predicted isoelectric point of 5.85. The sequence identity between L. donovani ALR (LdALR) and mammals and plants is only 36-44%. The ORF is a single copy gene. A protein with a molecular mass that matched the estimated approximately 74 kDa according to the amino acid composition of LdALR with a maltose binding tag present at its N-terminal end was induced by heterologous expression of LdALR in Escherichia coli. In the presence of glutathione, recombinant LdALR reduced methylglyoxal with a K(m) of approximately 112 microM. Comparative structural analysis of the human ALR structure with LdALR model suggests that the active site anchoring the N-terminal end of the glutathione is highly conserved. However, the C-terminal end of the glutathione backbone is expected to be exposed in LdALR, as the residues anchoring the C-terminal end of the glutathione backbone come from the three loop regions in human, which are apparently shortened in the LdALR structure. Thus, the computational analysis provides clues about the expected mode of glutathione binding and its interactions with the protein. This is the first report of the role of an ALR in the metabolic disposal of methylglyoxal in L. donovani and of thiol binding to a kinetoplastid aldose reductase.
Rath
Jyoti
J
School of Life sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
Gowri
V S
VS
Chauhan
Swati C
SC
Padmanabhan
Prasad K
PK
Srinivasan
N
N
Madhubala
Rentala
R
D000449:Aldehyde Reductase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D015139:Blotting, Southern; D004926:Escherichia coli; D018503:Genome, Protozoan / Q000235:genetics; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008658:Inactivation, Metabolic; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011765:Pyruvaldehyde / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D011765:Pyruvaldehyde; D000449:Aldehyde Reductase; D005978:Glutathione