Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

Abstract

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD+ salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from S. cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction where ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogs acting as inhibitors or substrates were examined revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary 15N and 13C kinetic isotope effects (KIE) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was observed indicative of a higher commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism is discussed involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid. These results will aid design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

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

“…In order to study the particular ionization state of catalytic residues in UbNic (D25, K111 and C155), the effect of pH on the k cat values was determined at a saturating nicotinamide concentration over a wide pH range (pH 4.5–10.0) ( Fig 5 ). The pH-dependence of k cat showed two catalytically relevant protonation equilibria for the enzyme substrate complex, one with an apparent p K es1 value of 4.2 ± 0.1, which must be unprotonated to be catalytically active (C155) and another p K es2 with a value of 8.6 ± 0.2 that must be protonated for activity (K111), as previously described for Saccharomyces cerevisiae nicotinamidase with pyrazinamide as substrate [ 29 ]. However, the difference, apart from the substrate used, is that UbNic showed a higher k cat in the alkaline pH range than in the acid one.…”

“…Interestingly, UbNic, PolyNic and PhNic are situated in a different clade from that formed by thermophilic archaeal nicotinamidases, which includes that from Acidilobus saccharovorans [ 16 ] ( Fig 2 , green). Other already described nicotinamidases are distributed in different clades throughout the phylogenetic tree, including a clade for yeasts ( Fig 2 , yellow) and nematodes ( Fig 2 , blue), where Saccharomyces cerevisiae and Caenorhabditis elegans nicotinamidases are included [ 18 , 24 , 26 , 28 , 29 , 56 ], respectively. The nicotinamidases from pathogenic microorganisms are distributed in three different clades, one including those of Borrelia burgdorferi , Leishmania infantum , Acinetobacter baumanii and the recently described nicotinamidase from Riemerella anatipestifer [ 4 – 6 , 18 , 20 ] ( Fig 2 , orange); and the others containing those of Mycobacterium tuberculosis [ 31 – 33 ] ( Fig 2 , red), and Streptococcus pneumoniae [ 18 , 34 ] ( Fig 2 , purple), respectively.…”

“…Nicotinaldehydes have been described as good competitive inhibitors for several nicotinamidases [ 11 , 18 , 29 , 31 , 34 ]. Among them, nicotinaldehyde and 5-bromo-nicotinaldehyde were the most potent and were used to show the tetrahedral thio-hemiacetal complex formed with the catalytic cysteine in SpNic during catalysis [ 34 ].…”

“…The effect of pH and pH-stability toward NAM was determined at 37°C in 100 mM sodium acetate (pH 4.0–5.0), sodium phosphate (pH 6.0–7.3), Tris-HCl (pH 8.0), glycine (pH 9.0–10.0) and CAPS (pH 11) or in 100 mM Citric-Hepes-CHES continuous buffer. In addition, the kinetic parameters were measured in the pH range 4.0–10.0 using buffers of constant ionic strength [ 29 , 46 ]. In this case, TBA buffer (50 mM Tris, 50 mM BisTris, 100 mM sodium acetate) was used in the pH range 4.0–8.5, and ATE buffer (100 mM ACES, 52 mM Tris, 52 mM ethanolamine) was used in the pH range 8.0–10.0.…”

“…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.…”