Abstract
Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km, values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His 138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the knowledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 279-285 |
| Number of pages | 7 |
| Journal | Biochemical Journal |
| Volume | 401 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2007 |
| Externally published | Yes |
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Keywords
- Chagas disease
- Pentose phosphate pathway
- Reaction mechanism
- Ribose 5-phosphate isomerase (Rpi)
- Trypanosoma cruzi
- Type B ribose 5-phosphate isomerase (RpiB)
ASJC Scopus subject areas
- Biochemistry
Cite this
Ribose 5-phosphate isomerase type B from Trypanosoma cruzi : Kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism. / Stern, Ana L.; Burgos, Emmanuel S.; Salmon, Laurent; Cazzulo, Juan J.
In: Biochemical Journal, Vol. 401, No. 1, 01.01.2007, p. 279-285.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Ribose 5-phosphate isomerase type B from Trypanosoma cruzi
T2 - Kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism
AU - Stern, Ana L.
AU - Burgos, Emmanuel S.
AU - Salmon, Laurent
AU - Cazzulo, Juan J.
PY - 2007/1/1
Y1 - 2007/1/1
N2 - Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km, values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His 138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the knowledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.
AB - Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km, values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His 138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the knowledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.
KW - Chagas disease
KW - Pentose phosphate pathway
KW - Reaction mechanism
KW - Ribose 5-phosphate isomerase (Rpi)
KW - Trypanosoma cruzi
KW - Type B ribose 5-phosphate isomerase (RpiB)
UR - http://www.scopus.com/inward/record.url?scp=33846318571&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33846318571&partnerID=8YFLogxK
U2 - 10.1042/BJ20061049
DO - 10.1042/BJ20061049
M3 - Article
C2 - 16981853
AN - SCOPUS:33846318571
VL - 401
SP - 279
EP - 285
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 1
ER -