Angiulli 2015 Biochim Biophys Acta
|Angiulli G, Lantella A, Forte E, Angelucci F, Colotti G, Ilari A, Malatesta F (2015) Leishmania infantum trypanothione reductase is a promiscuous enzyme carrying an NADPH:O2 oxidoreductase activity shared by glutathione reductase. Biochim Biophys Acta 1850:1891-7.|
Abstract: BACKGROUND: Leishmania infantum is a protozoan of the trypanosomatid family causing visceral leishmaniasis. Leishmania parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS2), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival.
METHODS: The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements.
RESULTS: The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the KM for NADPH decreases six times compared to the value obtained by adding TS2 as last reagent to start the reaction (1.9 vs. 12 μM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O2 to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O2 oxidoreductase activity is shared by Saccharomyces cerevisiae glutathione reductase (GR).
CONCLUSIONS: TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water.
GENERAL SIGNIFICANCE: TR and GR are promiscuous enzymes.
• Keywords: Electron transfer, Glutathione reductase, O2 reduction, Promiscuous enzymes, Sulfenic acid, Trypanothione reductase
• O2k-Network Lab: IT Roma Sarti P
Labels: MiParea: Instruments;methods
Enzymatic assay of trypanothione reductase