title: Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.
abstract: Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.
authors.affiliation: Department of Chemistry, State University of Ponta Grossa, 84030-900, Ponta Grossa, PR, Brazil.
authors.forename: Agnes Thiane Pereira
authors.initials: ATP
authors.lastname: Machado
authors.affiliation: Department of Education, Federal Technological University of Paraná, 84016-210, Ponta Grossa, PR, Brazil.
authors.forename: Marcio
authors.initials: M
authors.lastname: Silva
authors.affiliation: Department of Chemistry, State University of Ponta Grossa, 84030-900, Ponta Grossa, PR, Brazil. Electronic address: iulek@uepg.br.
authors.forename: Jorge
authors.initials: J
authors.lastname: Iulek
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009253:Naegleria / Q000201:enzymology / Q000235:genetics*; D000072417:Protein Domains; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Crystallization; Enzyme kinetics; Glyceraldehyde-3-phosphate dehydrogenase; Naegleria gruberi