title: Leishmania donovani trypanothione reductase: role of urea and guanidine hydrochloride in modulation of functional and structural properties.
abstract: Trypanothione reductase [TR], an NADPH-dependent disulfide oxidoreductase, unique to kinetoplastid parasites including Trypanosoma and Leishmania, is a validated target for the design of improved drugs. TR is a stable homodimer with a FAD molecule tightly bound to each subunit. In this paper, structure, function, stability properties and cofactor protein interactions of recombinant TR from Leishmania donovani were investigated under equilibrium unfolding/denaturing conditions. Urea induced unfolding was non-reductive in nature and led to the formation of partially folded intermediate. This intermediate species lacks catalytic activity and characteristic conformation of native LdTR but has significant secondary structure and could be partially reactivated. Guanidine hydrochloride-induced irreversible denaturation was marked by the presence of molten globule intermediate. Reactivation and cross-linking experiments clearly demonstrated that the loss of activity at lower denaturant concentrations was not coincided by dimer dissociation or structural unfolding. The studies demonstrate that functional conformation and stability are largely governed by ionic interactions and active site disulfide plays a vital role in maintaining functional conformation. The results obtained from this study provide intriguing insight into the possible mechanism/s of modulation of structure, function and stability of LdTR induced by the cationic, guanidine hydrochloride and the neutral denaturant, urea.
authors: Division of Biochemistry, Central Drug Research Institute, Lucknow-226001, India.
authors: Smita
authors: S
authors: Rai
authors: Upendra Nath
authors: UN
authors: Dwivedi
authors: Neena
authors: N
authors: Goyal
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D019281:Dimerization; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D019791:Guanidine; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea
chemical_list: D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; D014364:Tryptophan; D014508:Urea; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D019791:Guanidine