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

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Nicotinamide mononucleotide adenylyltransferases (NMNAT) synthesize NAD+.
• Existing NMNAT structures are from bacteria, archaea, and eukaryotes, but not thermophilic fungi.
• NAD+ is crucial for cellular metabolism.

Purpose of the Study:

• To elucidate the structure of NMNAT from the thermophilic fungus Chaetomium thermophilum (CtNMNAT).
• To characterize the enzymatic activity, stability, and kinetic properties of CtNMNAT.
• To explore the potential industrial applications of CtNMNAT in NAD+ biosynthesis.

Main Methods:

• X-ray crystallography was used to determine the 3D structure of CtNMNAT at 2.10 Å resolution.
• Enzyme activity assays were performed to assess catalytic efficiency and cofactor requirements.
• Biophysical methods were employed to evaluate thermostability and optimal reaction conditions (pH, temperature).

Main Results:

• The crystal structure of CtNMNAT revealed a nicotinamide mononucleotide (NMN) molecule in the active site.
• CtNMNAT exhibits high catalytic activity (kcat/KM = 180 s⁻¹ mM⁻¹) and good thermostability (half-life of 25.9 h at 50 °C).
• Optimal activity was observed at pH 6.0 and 60 °C, with a high space-time yield of 72.0 g L⁻¹ d⁻¹ for NAD+ production.

Conclusions:

• The structure of CtNMNAT provides insights into NAD+ biosynthesis in thermophilic fungi.
• CtNMNAT demonstrates significant potential for industrial biocatalytic synthesis of NAD+ due to its high activity and stability.
• This study lays the groundwork for further research into CtNMNAT's role in NAD+ metabolism and its biotechnological applications.