Novel Calcium-Binding Motif Stabilizes and Increases the Activity of Aspergillus fumigatus Ecto-NADase

Biochemistry. 2023 Nov 21;62(22):3293-3302. doi: 10.1021/acs.biochem.3c00360. Epub 2023 Nov 7.


Nicotinamide adenine dinucleotide (NAD) is an essential molecule in all kingdoms of life, mediating energy metabolism and cellular signaling. Recently, a new class of highly active fungal surface NADases was discovered. The enzyme from the opportunistic human pathogen Aspergillus fumigatus was thoroughly characterized. It harbors a catalytic domain that resembles that of the tuberculosis necrotizing toxin from Mycobacterium tuberculosis, which efficiently cleaves NAD+ to nicotinamide and ADP-ribose, thereby depleting the dinucleotide pool. Of note, the A. fumigatus NADase has an additional Ca2+-binding motif at the C-terminus of the protein. Despite the presence of NADases in several fungal divisions, the Ca2+-binding motif is uniquely found in the Eurotiales order, which contains species that have immense health and economic impacts on humans. To identify the potential roles of the metal ion-binding site in catalysis or protein stability, we generated and characterized A. fumigatus NADase variants lacking the ability to bind calcium. X-ray crystallographic analyses revealed that the mutation causes a drastic and dynamic structural rearrangement of the homodimer, resulting in decreased thermal stability. Even though the calcium-binding site is at a long distance from the catalytic center, the structural reorganization upon the loss of calcium binding allosterically alters the active site, thereby negatively affecting NAD-glycohydrolase activity. Together, these findings reveal that this unique calcium-binding site affects the protein fold, stabilizing the dimeric structure, but also mediates long-range effects resulting in an increased catalytic rate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus fumigatus / genetics
  • Aspergillus fumigatus / metabolism
  • Calcium
  • Humans
  • NAD* / metabolism
  • NAD+ Nucleosidase* / chemistry
  • NAD+ Nucleosidase* / genetics
  • NAD+ Nucleosidase* / metabolism
  • Niacinamide


  • NAD+ Nucleosidase
  • NAD
  • Calcium
  • Niacinamide