Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli.
Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.
Mittal
Mukul K
MK
Division of Biochemistry, Central Drug Research Institute, Lucknow, India.
Misra
Smita
S
Owais
Mohammad
M
Goyal
Neena
N
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010802:Phylogeny; D016415:Sequence Alignment; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism
C044809:trypanothione; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005978:Glutathione; D013095:Spermidine