Thermodynamics, functional and structural characterization of inosine-uridine nucleoside hydrolase from Leishmania braziliensisElectronic supplementary information (ESI) available. See DOI: 10.1039/c7ra07268f
Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries with the highest incidence of cutaneous and mucocutaneous forms of leishmaniasis caused mainly by Leishmania braziliensis . The first-line drugs used in the treatment of leishmaniasis have se...
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Main Authors | , , , , , , , , , , , , , , , , |
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Format | Journal Article |
Published |
17.10.2017
|
Online Access | Get full text |
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Summary: | Leishmaniasis is considered one of the main endemic diseases in the world, and Brazil is among the countries with the highest incidence of cutaneous and mucocutaneous forms of leishmaniasis caused mainly by
Leishmania braziliensis
. The first-line drugs used in the treatment of leishmaniasis have several limitations: parenteral administration, long duration of treatment, and serious toxicity. One key metabolic characteristic of these parasites is the lack of a
de novo
purine biosynthesis pathway, making them auxotrophic to purines. Accordingly, they rely solely on the purine salvage pathway for nucleotide synthesis. A better understanding of the purine salvage pathway can reveal details of the biology of
L. braziliensis
that could, in turn, be used to develop new strategies to combat this parasite. The inosine-uridine nucleoside hydrolase from
L. braziliensis
(
Lb
IU-NH) plays an important role in the salvage process and is an attractive drug target as there is no similar catalytic activity in mammals. Here is described cloning, heterologous protein expression, and a three-step purification protocol that yielded homogenous recombinant protein. The determination of
Lb
IU-NH steady-state kinetic constants for inosine, adenosine, cytidine, uridine and
p
-nitrophenyl β-
d
-ribofuranoside is also reported. These data suggest that
Lb
IU-NH displays characteristics of a nonspecific hydrolase. The thermodynamic profile suggests that
d
-ribose can bind to free enzyme with favorable enthalpic (Δ
H
) and entropic (Δ
S
) contributions. Thermodynamic activation parameters (
E
a
, Δ
G
#
, Δ
S
#
, Δ
H
#
) for the
Lb
IU-NH-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, and pH-rate profiles are also presented. In addition, the crystal structure of
Lb
IU-NH in complex with β-
d
-ribose and Ca
2+
at 1.5 Å resolution is described.
Inosine-uridine nucleoside hydrolase from
Leishmania braziliensis
is a nonspecific enzyme that contains a disulfide bond not needed for tetramer stabilization. |
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Bibliography: | 10.1039/c7ra07268f Electronic supplementary information (ESI) available. See DOI |
ISSN: | 2046-2069 |
DOI: | 10.1039/c7ra07268f |