scholarly journals Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

2006 ◽  
Vol 401 (1) ◽  
pp. 279-285 ◽  
Author(s):  
Ana L. Stern ◽  
Emmanuel Burgos ◽  
Laurent Salmon ◽  
Juan J. Cazzulo
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Type B ◽  
Nucleotide Synthesis ◽  
Gene Coding ◽  
Genes Encoding

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-phos-phate) 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 competi-tively, 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 His138, 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 know-ledge 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.

scholarly journals The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

2007 ◽  
Vol 79 (4) ◽  
pp. 649-663 ◽  
Author(s):  
Mariana Igoillo-Esteve ◽  
Dante Maugeri ◽  
Ana L. Stern ◽  
Paula Beluardi ◽  
Juan J. Cazzulo
Keyword(s):  
Oxidative Stress ◽  
Trypanosoma Cruzi ◽  
Pentose Phosphate Pathway ◽  
Single Copy ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Haploid Genome ◽  
Salt Bridges ◽  
Phosphogluconate Dehydrogenase ◽  
Genes Encoding

Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

scholarly journals Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

1997 ◽  
Vol 321 (3) ◽  
pp. 609-614 ◽  
Author(s):  
Luc BERTRAND ◽  
Didier VERTOMMEN ◽  
Ernest FEYTMANS ◽  
Attilio Di PIETRO ◽  
Mark H. RIDER ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Magnesium Citrate ◽  
Phosphate Binding ◽  
Conserved Sequence ◽  
Kinase Mutation

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg-138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

scholarly journals Biochemical and genetic characterization of Trypanosoma cruzi N-myristoyltransferase

2014 ◽  
Vol 459 (2) ◽  
pp. 323-332 ◽  
Author(s):  
Adam J. Roberts ◽  
Leah S. Torrie ◽  
Susan Wyllie ◽  
Alan H. Fairlamb
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Genetic Characterization ◽  
Kinetic Properties

The present study shows that N-myristoyltransferase is essential for growth of Trypanosoma cruzi, the parasite responsible for Chagas’ disease. The kinetic properties of the enzyme are described along with evidence that growth is specifically inhibited by blocking N-myristoylation in the parasite.

scholarly journals Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on SubstrateKm

2001 ◽  
Vol 45 (3) ◽  
pp. 805-809 ◽  
Author(s):  
Ylva Haasum ◽  
Katrin Ström ◽  
Rahma Wehelie ◽  
Vicki Luna ◽  
Marilyn C. Roberts ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Washington State ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Directed Mutagenesis ◽  
Aminobenzoic Acid ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Gene Coding ◽  
Sulfonamide Resistance

ABSTRACT Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniaeisolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile66 and Glu67 in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Ki for sulfathiazole but changed the Km for p-aminobenzoic acid only 2.5-fold and did not significantly change theKm for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61 had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

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