Characterization of Nicotinamidases: Steady State Kinetic Parameters, Classwide Inhibition by Nicotinaldehydes, and Catalytic Mechanism

Abstract

Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast and invertebrates but there are none found in mammals. Although recent structural work has improved understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data shows that nicotinamidases are required for growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans regulate lifespan in their respective organisms, consistent with proposed roles in the regulation of NAD+ metabolism and organismal aging. In this manuscript, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, S. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme Disease) and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state kcat values typically exceeding 1 s−1. The Km values for nicotinamide are low and are in the range from 2 – 110 µM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low µM to low nM range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex which is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyse exchange of 18O into the carboxy oxygens of nicotinic acid with 18O-water. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid 18O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion and the intermediacy of a thioester intermediate.

Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD+ Concentration

“…We cannot rule out the possibility that INAM is directly metabolized by yeast cells to produce NAD ϩ when present at high millimolar concentrations. Pnc1-mediated nicotinamidase activity is not detectable in vitro with 500 M INAM as a substrate (55), but some activity was detectable in vitro above 25 mM (Fig. 6C).…”

Comparative Metabolomic Profiling Reveals That Dysregulated Glycolysis Stemming from Lack of Salvage NAD+ Biosynthesis Impairs Reproductive Development in Caenorhabditis elegans

“…The C. elegans genome encodes a second nicotinamidase called PNC-2. Kinetically, PNC-2 is Ͻ10% as efficient as PNC-1 (34). Moreover, RNAi of pnc-2 did not produce any obvious phenotypes in wild type or any synthetic phenotypes in pnc-1(pk9605) nor did it exacerbate any pnc-1(pk9605) phenotypes (Ref.…”

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

“…The alignment also revealed that PolyNic contained all the conserved catalytic triad residues of the cysteine-hydrolases family ( Figure 5 , filled circles): a catalytic cysteine at position 150 (C150, PolyNic numbering), an aspartate at position 25 (D25) and a lysine at position 111 (K111) (French et al, 2010b; Sanchez-Carron et al, 2013; Ion et al, 2014; Sheng and Liu, 2014). Furthermore, PolyNic showed the characteristic cis -peptide bond ( Figure 5 , positions L145 and A146, filled squares), which is invariably preceded by a conserved glycine (G144), and the conserved specific metal ion binding, which includes one aspartate (D67) and two histidines (H69 and H86) ( Figure 5 , filled triangles).…”

“…PolyNic exhibited its maximum activity at a temperature of 50°C ( Figure 6B ), which is 5 to 20°C higher than those described for other nicotinamidases (Pardee et al, 1971; Yan and Sloan, 1987; Zhang et al, 2008; Sanchez-Carron et al, 2013). However, and for comparison purposes, kinetic parameters were carried out at pH 7.3 and 37°C, as generally accepted in the bibliography (Zhang et al, 2008; French et al, 2010b; Sanchez-Carron et al, 2013). …”

“…Nicotinamidases (EC 3.5.1.19) are key metal-dependent amidohydrolases in NAD + metabolism of multiple species of archaea (Stekhanova et al, 2014), bacteria (Frothingham et al, 1996; Boshoff and Mizrahi, 1998; Zhang et al, 2008; French et al, 2010b; Sanchez-Carron et al, 2013), yeast (Joshi and Handler, 1962; Hu et al, 2007), protozoa (Zerez et al, 1990; Gazanion et al, 2011) and plants (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 also 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 then recycled to NAD + (Opitz and Heiland, 2015).…”