A thioredoxin reductase-class of disulphide reductase in the protozoan parasite Giardia duodenalis.
We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent (Km = 8 microM) reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called "glutathione-induced thiol-reductase activity' previously observed in G. duodenalis.
Brown
D M
DM
Queensland Institute of Medical Research, Bancroft Centre, Brisbane, Queensland, Australia.
Upcroft
J A
JA
Upcroft
P
P
D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005872:Giardia / Q000201:enzymology*; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009579:Nitrobenzoates / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*
D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; C011136:thionitrobenzoic acid; D009249:NADP; D004228:Dithionitrobenzoic Acid; D005979:Glutathione Peroxidase; D009247:NADH, NADPH Oxidoreductases; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase