Herein, we describe a novel enzymatic cycling method to measure nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), which are precursors of NAD biosynthesis. A gene encoding an NMN adenylyltransferase (NMNAT, EC 22.214.171.124) homologue was identified in Thermus thermophilus HB8. The gene from T. thermophilus (TtNMNAT) was engineered for expression in Escherichia coli and the recombinant enzyme found to be stable, retaining full activity after incubation for 45 min at 70 C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27 mM, respectively, with a Vmax value of 60.3 mmoL/min/mg. TtNMNAT was successfully applied to the colorimetric NMN or NaMN assays, which employed (i) adenylation of NMN to NAD by TtNMNAT or adenylation of NaMN to deamido-NAD (NaAD) by TtNMNAT followed by amidation of NaAD to NAD by NAD synthetase (NADS, EC 126.96.36.199) and (ii) an NAD cycling reaction using 12a-hydroxysteroid dehydrogenase (12a-HSD, EC 188.8.131.52) and diaphorase (DI, EC 184.108.40.206) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5 mM (12.2 nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.
NAD intermediates, including nicotinamide mononucleotide (NMN) and nicotinic acid mononucleotide (NaMN), may function as signaling molecules to regulate NAD homeostasis, which has increased the importance of developing a robust assay for these compounds. Aberrant NAD metabolism has been implicated in many metabolic- and age-associated diseases such as cancer and diabetes (1,2). NMN along with riboside have been reported to improve health span in mouse models of muscle aging and cognitive decline (3,4). Although the mechanism of action is unclear, NAD precursors may be involved in the activation of sirtuin NAD-dependent proteins (2,5). In addition, the potential of NMN to act as a prognostic marker of early stage diabetic nephropathy has been proposed (6,7). NMN is currently available on the market as a nutraceutical, but its safety and effect on human physiology is unknown. The purpose of this study was to develop a convenient and sensitive assay for NMN and NaMN that can be applied to automatic clinical analyzers.
Here, an NAD cycling method was employed for the measure- ment of NMN and NaMN, so that the sensitivity and measurement range could be readily adjusted. The assay required adenylyl- transferase (NMNAT, EC 220.127.116.11), which could adenylate the analy- tes. NMNAT is a ubiquitous enzyme that functions to maintain NAD homeostasis and is therefore essential for proper cellular function. MNAT derived from Saccharomyces cerevisiae was previously tested for measuring PPi, but was too unstable to be used in the NMN and NaMN assay procedure. However, we reasoned that NMNAT from Thermus thermophilus HB8 (TtNMNAT) might be a more stable enzyme for the assay.
MATERIALS AND METHODS
Materials The pET21a vector was obtained from Novagen (Madison, WI, USA) and expression trials in Escherichia coli were performed according to the manufacturer’s instructions. E. coli strain BL21(DE3) was purchased from Stratagene (La Jolla, CA, USA). NAD synthetase from Geobacillus stearothermophilus (NADS, EC 18.104.22.168), 12a-hydroxysteroid dehydrogenase from Bacillus sphaericus (12a-HSD, EC 22.214.171.124) and diaphorase from Bacillus megaterium (DI, EC 126.96.36.199) were from Asahi Kasei Pharma (Tokyo, Japan). Fresh human sera and urine were purchased from BizCom Japan (Tokyo, Japan).
All other chemicals used in this study were purchased from Dojindo Labora- tories (Kumamoto, Japan), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), or SigmaeAldrich Japan Co., LLC. (Tokyo, Japan).
Cloning of the gene encoding TtNMNAT A synthetic gene encoding nico- tinamide mononucleotide adenylyltransferase (NMNAT, EC 188.8.131.52) from T. thermophilus HB8 (TTHA1780) was synthesized by GenScript (Piscataway, NJ, USA). The synthetic gene (0.56-kbp) contained a unique NdeI and HindIII restriction site at the 50 and 30 end, respectively. The gene was cloned into the corresponding sites in the pET21a vector to yield TtNMNAT/pET21a.
Overproduction and purification of TtNMNAT E. coli BL21(DE3) cells were transformed with TtNMNAT/pET21a, and the transformants selected by growth on LB agar supplemented with ampicillin (50 mg/ml). A single colony was then picked and grown in 20 L of Overnight Express Instant TB Medium (Novagen) containing 50 mg/ml ampicillin for 24 h at 30 C. Cells expressing TtNMNAT, were harvested by centrifugation, suspended in 10 mM TriseHCl (pH 8.5), and then lysed by addition of 0.5% lysozyme, 1 mM EDTA, 0.05% Triton X-100 followed by a 1 h incubation at 37 C. After removing the cell debris by centrifugation for 30 min at 5000 g, the resultant lysate was heated for 30 min at 70 C, and the denatured proteins removed by centrifugation for 30 min at 5000 g. The supernatant was then loaded onto a 3 L of Q Sepharose Big Beads (Q sep. BB) column (BPG100, GE Healthcare) pre-equilibrated with 10 mM TriseHCl (pH 8.5). After washing the column with the equilibration buffer, the bound protein was eluted with a linear gradient of 0e0.5 M KCl in the same buffer. Active fractions were concentrated using a 30-kDa centrifugal filter device (Millipore, Bedford, MA, USA) and solid KCl was added to achieve a 3 M concentration. The protein solution was applied to a 500 mL Phenyl Sepharose 6 Fast Flow (Phenyl sep. ff) column (High Sub) (XK50, GE Healthcare) pre-equilibrated with 10 mM TriseHCl containing 3 M KCl buffer (pH 7.5). The column was then washed with equilibration buffer, and the bound protein eluted with a linear gradient of 3 to 0 M KCl in the same buffer. Fractions containing TtNMNAT activity were pooled, concentrated using a 30-kDa centrifugal filter device, and dialyzed against 10 mM TriseHCl buffer (pH 8.5). The protein solution was applied to a 250 mL Q Sepharose High Performance (Q sep. HP) column (XK50, GE Healthcare) pre-equilibrated with 10 mM TriseHCl buffer (pH 8.5). The column was then washed with equilibration buffer, and the protein eluted with a linear gradient of 0e0.5 M KCl in the same buffer. Active fractions were pooled and then concentrated using a 30-kDa centrifugal filter device. The sample was desalted by gel filtration chromatography using a Sephadex G-25 Superfine column (GE Healthcare) pre-equilibrated with 10 mM TriseHCl (pH 7.5). The entire purification procedure was performed at room temperature (<25 C).
Enzyme assays Unless otherwise specified, TtNMNAT activity was routinely determined in a continuous reaction entailing (i) ATP-dependent NAD formation from NMN and (ii) oxidation of cholic acid to 3-oxocholic acid by 12a-HSD with concomitant conversion of NAD to NADH. Standard reaction mixtures comprised 100 mM HEPES-NaOH (pH 8.0), 1 mM NMN, 1 mM ATP, 4 mM NiCl2, 20 U/mL 12a- HSD and 4 mM cholic acid in a total volume of 150 mL. The rate of NADH oxidation at 37 C was monitored spectrophotometrically at 340 nm with d 1⁄4 1 cm and ε340 1⁄4 6.22 mM 1 cm 1. One unit (U) of enzyme was defined as the amount of enzyme forming 1 mmol of NADH.
Cycling rate constant The cycling rate constant (kc; in rotations per minute) was calculated using the following equation (8):
where DAbs is the absorbance change per minute, EV is the volume of the enzyme solution (in mL) added to the reaction mixture, εcoeffi is the molar extinction coef- ficient, and Csub is the substrate concentration.
Molecular mass The molecular mass of purified TtNMNAT in solution was determined by gel filtration chromatography on a Superdex 200 column pre- equilibrated with 50 mM potassium phosphate buffer (pH 7.0) containing 150 mM NaCl (in the absence of reducing agents). The molecular mass standards used to calibrate the column were: glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), myokinase (32 kDa) and cytochrome c (12.4 kDa).
MichaeliseMenten kinetics For Km determinations, 0.1e5 mM NMN and 0.1e5 mM ATP were used. The NiCl2 concentration was the same as the ATP concentration.
NMN or NaMN assay NMN was assayed by measuring the increase in absorbance at 450 nm with the production of reduced WST-8 (water-soluble tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo- phenyl)-2H-tetrazolium, Dojindo Laboratories). Reaction mixture R-1 contained 200 mM HEPES-NaOH (pH 8.0), 1 mM ATP, 4 mM NiCl2, 0.4 mM WST-8, 10 U/mL TtNMNAT, and 30 U/mL 12a-HSD. Reaction mixture R-2 contained 200 mM HEPES-NaOH (pH 8.0), 24 mM cholate and 180 U/mL DI. An automated assay for NMN was performed using a model 7170 Hitachi automatic clinical analyzer (Hitachi, Tokyo). In the NMN assay, 150 mL of R-1 was incubated with 5 mL samples for 5 min at 37 C, after which 50 mL of R-2 was added. After a further incubation for 3.5 min, reduced WST-8 formation was measured for 1 min based on the absorbance increase at 450 nm (ε450 1⁄4 30.0 mM 1 cm 1). The assay mode was Rate-A (rate assay).
NaMN was also assayed by measuring the increase in absorbance at 450 nm with the production of reduced WST-8. Reaction mixture R-10 contained 200 mM HEPES- NaOH (pH 8.0), 2 mM ATP, 1.5 mM MgCl2, 4 mM NiCl2, 0.4 mM WST-8, 10 U/mL TtNMNAT, 30 U/mL 12a-HSD, 2 mM NH4Cl, and 5 U/mL NADS. Reaction mixture R-2 and the automated assay method were the same as those used for NMN determination.