title: In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.
abstract: Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Curitiba, Brazil.
authors.forename: Kerly
authors.initials: K
authors.lastname: Laskoski
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Curitiba, Brazil.
authors.forename: Adrian R S
authors.initials: AR
authors.lastname: Santos
authors.affiliation: Departamento de Genética, UFPR, Curitiba, PR, Brazil.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Bonatto
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Curitiba, Brazil.
authors.forename: Fábio O
authors.initials: FO
authors.lastname: Pedrosa
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Curitiba, Brazil.
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: Souza
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Curitiba, Brazil. luciano.huergo@gmail.com.|Setor Litoral, UFPR, Matinhos, Brazil. luciano.huergo@gmail.com.
authors.forename: Luciano F
authors.initials: LF
authors.lastname: Huergo
mesh_terms: D019732:Amide Synthases / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D005973:Glutamine / Q000378:metabolism; D042803:Herbaspirillum / Q000201:enzymology*; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism
chemical_list: D005973:Glutamine; D009243:NAD; C018348:nicotinic acid adenine dinucleotide; D019732:Amide Synthases; C038479:NAD+ synthase; D009584:Nitrogen
keywords: Enzyme; Glutaminase; NAD+