Biochemical and structural characterization of a novel nicotinamide mononucleotide adenylyltransferase from thermophilic fungi Chaetomium thermophilum.

Nicotinamide mononucleotide adenylyltransferases (NMNAT EC: 2.7.7.1) play pivotal roles in synthesis of nicotinamide adenine dinucleotide (NAD) through catalyzing the reaction of nicotinamide mononucleotide (NMN) with adenosine triphosphate (ATP). To date, multiple crystal structures of NMNAT originated from bacteria, archaea, and eukaryote have been resolved. However, none structure of NMNAT from thermophilic fungi was elucidated. Here we report the structure of nicotinamide mononucleotide adenylyltransferase (CtNMNAT) from thermophilic fungi Chaetomium thermophilum at a resolution of 2.10 Å. The enzyme crystals were in space group of P222 with two monomers per asymmetric unit. A nicotinamide mononucleotide (NMN) molecule was identified in the active pocket. Enzyme activity assays confirmed that CtNMNAT has a relatively high activity and good thermostability. The pH and temperature optima of CtNMNAT were pH 6.0 and 60 °C. CtNMNAT was stable at 50 °C with a half-life of 25.9 h. The specificity constant k/K of CtNMNAT toward NMN was 180 s mM 28.5 mM NAD was produced when using CtNMNAT as catalyst with a space-time yield of 72.0 g L d. This work provides a starting point for further investigation into the role of CtNMNAT in the NAD  metabolism of Chaetomium thermophilum and useful guidance for industrial application of CtNMNAT in the biocatalytic synthesis of NAD.