Abstract
Nicotinamidases catalyze the hydrolysis of the amide bond in nicotinamide (NAM) to produce ammonia and nicotinic acid (NA). These enzymes are an essential component of the NAD+ salvage pathway and are implicated in the viability of several pathogenic organisms. Its absence in humans makes them a promising drug target. In addition, although they are key analytical biocatalysts for screening modulators in relevant biomedical enzymes, such as sirtuins and poly-ADP-ribosyltransferases, no commercial sources are available. Surprisingly, the finding of an affordable source of nicotinamidase from metagenomic libraries is hindered by the absence of a suitable and fast screening method. In this manuscript, we describe the development of two new whole-cell methods using the chemical property of one of the products formed in the enzymatic reaction (pyrazinoic or NA) to form colored complexes with stable iron salts, such as ammonium ferrous sulfate or sodium nitroprusside (SNP). After optimization of the assay conditions, a fosmid polygenomic expression library obtained from deep-sea mesophilic bacteria was screened, discovering several positive clones with the ammonium ferrous sulfate method. Their quantitative rescreening with the SNP method allowed the finding of the first nicotinamidase with balanced catalytic efficiency toward NAM (nicotinamidase activity) and pyrazinamide (pyrazinamidase activity). Its biochemical characterization has also made possible the development of the first high-throughput whole-cell method for prescreening of new nicotinamidase inhibitors by the naked eye, saving time and costs in the design of future antimicrobial and antiparasitic agents.
Highlights
IntroductionNicotinamidase High-Throughput Functional Screening (Wang and Pichersky, 2007) that catalyze the hydrolysis of nicotinamide (NAM) (Figure 1A) and its analog pyrazinamide (PZA) (Figure 1B) to nicotinic acid (NA) or pyrazinoic acid (POA) and ammonia, respectively
At the highest concentration of Nicotinamidase activity is usually followed by reverse phase high-performance liquid chromatography (HPLC) using its natural substrate (NAM), or continuously monitored following one of the two products of the nicotinamidase reaction in a coupled assay with bovine or recombinant glutamate dehydrogenases (GDH) (French et al, 2010b; Sanchez-Carron et al, 2013)
This pro-drug is converted into pyrazinoic acid (POA) by Mycobacterium tuberculosis nicotinamidase (Kalinda and Aldrich, 2012)
Summary
Nicotinamidase High-Throughput Functional Screening (Wang and Pichersky, 2007) that catalyze the hydrolysis of nicotinamide (NAM) (Figure 1A) and its analog pyrazinamide (PZA) (Figure 1B) to nicotinic acid (NA) or pyrazinoic acid (POA) and ammonia, respectively They are present in many metazoans such as Drosophila melanogaster (Balan et al, 2008) and Caenorhabditis elegans (van der Horst et al, 2007), but absent in mammals, since they alternatively use NAM phosphoribosyltransferase (NAMPT) to convert NAM directly to NAM mononucleotide (NMN), which is recycled to NAD+ (Opitz and Heiland, 2015). It is not surprising that the majority of metagenome-derived enzymes that have been biochemically characterized are mainly esterases/lipases and glycoside hydrolases (Lopez-Lopez et al, 2014; Sathya and Khan, 2014; Ufarte et al, 2015)
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