Limits...
Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi.

Niño CH, Forero-Baena N, Contreras LE, Sánchez-Lancheros D, Figarella K, Ramírez MH - Mem. Inst. Oswaldo Cruz (2015)

Bottom Line: In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models.The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector.These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.

ABSTRACT
The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite's drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Show MeSH

Related in: MedlinePlus

multiple alignments of 16 nicotinamide mononucleotide adenylyltransferaseisozymes from phylogenetically divergent organisms. Sequences used were indexedand annotated in the UniProtKB database and are shown with their correspondingidentification code and the degree of conservation of the residues along thesequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660618&req=5

f01: multiple alignments of 16 nicotinamide mononucleotide adenylyltransferaseisozymes from phylogenetically divergent organisms. Sequences used were indexedand annotated in the UniProtKB database and are shown with their correspondingidentification code and the degree of conservation of the residues along thesequences.

Mentions: Identification of NMNAT candidate sequences in the genome of T. cruzi -Based on a multiple sequence alignment of NMNAT isozymes (Fig. 1), a consensus sequence of 244 residues was obtained. This sequence wascomposed of residues that were highly conserved among the initial sequences. Using thissequence, a search was performed on the genomes of four T. cruzistrains using the BLASTP algorithm. A candidate sequence was found for eachgenome, producing a total of four sequences with scores and significant p-values asshown in Table I; each of these genes encodes aprotein of 289 aa and 32 kDa from a coding region of 870 nucleotides. For all fourgenes, a biosynthetic function (predicted GO process) and a nucleotidyltransferaseactivity were predicted (predicted GO function).


Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi.

Niño CH, Forero-Baena N, Contreras LE, Sánchez-Lancheros D, Figarella K, Ramírez MH - Mem. Inst. Oswaldo Cruz (2015)

multiple alignments of 16 nicotinamide mononucleotide adenylyltransferaseisozymes from phylogenetically divergent organisms. Sequences used were indexedand annotated in the UniProtKB database and are shown with their correspondingidentification code and the degree of conservation of the residues along thesequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660618&req=5

f01: multiple alignments of 16 nicotinamide mononucleotide adenylyltransferaseisozymes from phylogenetically divergent organisms. Sequences used were indexedand annotated in the UniProtKB database and are shown with their correspondingidentification code and the degree of conservation of the residues along thesequences.
Mentions: Identification of NMNAT candidate sequences in the genome of T. cruzi -Based on a multiple sequence alignment of NMNAT isozymes (Fig. 1), a consensus sequence of 244 residues was obtained. This sequence wascomposed of residues that were highly conserved among the initial sequences. Using thissequence, a search was performed on the genomes of four T. cruzistrains using the BLASTP algorithm. A candidate sequence was found for eachgenome, producing a total of four sequences with scores and significant p-values asshown in Table I; each of these genes encodes aprotein of 289 aa and 32 kDa from a coding region of 870 nucleotides. For all fourgenes, a biosynthetic function (predicted GO process) and a nucleotidyltransferaseactivity were predicted (predicted GO function).

Bottom Line: In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models.The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector.These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.

ABSTRACT
The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite's drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Show MeSH
Related in: MedlinePlus