In vitro characterization of the NAD + synthetase NadE1 from Herbaspirillum seropedicae
- PDF / 621,676 Bytes
- 7 Pages / 595.276 x 790.866 pts Page_size
- 114 Downloads / 167 Views
ORIGINAL PAPER
In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae Kerly Laskoski1 · Adrian R. S. Santos1 · Ana C. Bonatto2 · Fábio O. Pedrosa1 · Emanuel M. Souza1 · Luciano F. Huergo1,3
Received: 23 October 2015 / Revised: 18 December 2015 / Accepted: 8 January 2016 © Springer-Verlag Berlin Heidelberg 2016
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
Communicated by Jorge Membrillo-Hernández. Kerly Laskoski and Adrian R. S. Santos have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00203-016-1190-z) contains supplementary material, which is available to authorized users. * Luciano F. Huergo [email protected] 1
Departamento de Bioquímica e Biologia Molecular, Curitiba, Brazil
2
Departamento de Genética, UFPR, Curitiba, PR, Brazil
3
Setor Litoral, UFPR, Matinhos, Brazil
kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor. Keywords NAD+ · Glutaminase · Enzyme
Introduction Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) act as important coenzymes in redox reactions. Apart from the coenzyme function, NAD+ acts as a substrate for a variety of NAD+-consuming enzymes such as ADP-ribosyl transferases, DNA ligase and sirtuins (Lin 2007; Bi et al. 2011). Therefore, NAD+ must be constantly replenished to keep homeostasis, and this is achieved by NAD+ de novo or salvage pathways (Gazzaniga et al. 2009). The last reaction step in de novo pathways and also in some of the NAD+ salvage pathways described to date is the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD+. This reaction is catalyzed by the ubiquitous enzymes named NAD synthetases or NadE. Given the key role played by NadE and its broad distribution, this enzyme has attracted considerable interest as a potential target for development of novel antibiotics (Bi et al. 2011). Indeed, controlled knockout of the Mycobacter
Data Loading...
