Abstract
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 2.7.7.1) 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 45min at 70°C. The Km values for NMN and ATP were calculated to be 0.263 and 1.27mM, respectively, with a Vmax value of 60.3μmoL/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 6.3.1.5) and (ii) an NAD cycling reaction using 12α-hydroxysteroid dehydrogenase (12α-HSD, EC 1.1.1.176) and diaphorase (DI, EC 1.6.99.3) to accumulate reduced WST-8. This enzymatic cycling method enabled detection of 0.5μM (12.2nM in the reaction mixture) NMN or NaMN in an automatic clinical analyzer.
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