Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries

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.

Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community

“…The phylogenetic analysis carried out with MEGA [ 54 ] showed that UbNic clusters in the same clade as the archaeal Pyrococcus horikoshii nicotinamidase [ 13 ] and the recently described polygenomic nicotinamidase PolyNic [ 11 ] ( Fig 2 , cyan). This clade contains a variety of sediment-growing and thermophilic microorganisms from both the Bacteria and the Archaea domains.…”

“…After sequencing, a putative nicotinamidase sequence was found in fosmid JFF054_F02. Then, the clone containing the latter fosmid was checked for nicotinamidase activity using the previously described pyrazinamide/ammonium ferrous sulfate method [ 11 ]. Briefly, after growing fosmids at 37°C overnight, their pellets were resuspended in a mixture of 20 mM pyrazinamide and 1% ammonium ferrous sulfate dissolved in MilliQ water, and incubated for 1 h at 37°C until an intense orange-red color appeared.…”

“…The first nicotinamidase activity was reported in 1952 [ 10 ], since when the activity of 24 more of these enzymes has been measured [ 6 , 11 – 37 ]. However, their catalytic efficiency toward their respective natural substrate, nicotinamide, has only been reported in 12 of them [ 11 , 12 , 14 , 16 – 18 , 29 , 31 ], and only in 6 of these also toward other nicotinamide analogs [ 11 , 16 , 17 , 20 , 29 , 31 ]. Given that more than 8300 nicotinamidase sequences derived from large-scale sequencing and metagenomics have been deposited in the UniProt database, the number of nicotinamidases described to date represents only a 0.2% of the total.…”

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…The hydrolytic activity of the recombinant enzymes was assayed qualitatively by testing 29 different substrates ( Figure 4A): N 4 -acylated (2'-deoxy)cytidines and cytosines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16); N 2 -acetylisocytosines (17); chromogenic substrates such as p-nitroacetanilide (18) and p-nitrobenzanilide (19); various p-nitrophenyl (pNP) esters (20)(21)(22)(23)(24); terephthalate derivatives bis(2-hydroxyethyl) terephthalate (26), dimethyl terephthalate (27), and monomethyl terephthalate (28); and the beta-lactam nitrocefin (29). Hydrolysis of chromogenic substrates (18)(19)(20)(21)(22)(23)(24)(25) was analysed spectrophotometrically at 405 nm, and that of other substrates was analysed spectrophotometrically at 240-320 nm and using TLC or HPLC-MS ( Figures S4-S10). All the reactions were monitored up to 24 h. The 8 hydrolases selected from the metagenomic libraries showed different activity profiles towards N-acylated nucleosides harbouring short, bulky aliphatic, aromatic, and heterocyclic groups ( Figure 4B).…”

“…Bacterial gene libraries, constructed from an environment intentionally enriched in N-acylhomoserine lactone-degrading bacteria [22], have been used as well. The acyl transfer activity of amidase in the presence of hydroxylamine, resulting in the spectrophotometric quantification of hydroxamic acid/iron(III) complexes, as well as the whole-cell methods that use the chemical properties of one of the products formed in the amidohydrolase enzymatic reaction to form coloured complexes with stable iron salts have been proposed [23,24]. A system that is based on pro-chromogenic substrates combined with an auxiliary enzyme [25] and an effective approach based on product detection-the product-induced gene expression (PIGEX) [26] system-have also been reported to be suitable for screening of amidohydrolases in metagenomes.…”