The crystal structure and mutational analysis of human NUDT9

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Human ADP-ribose pyrophosphatase (NUDT9) is a Nudix hydrolase involved in cellular detoxification.
• NUDT9 shares homology with the TRPM2 ion channel's catalytic domain, which possesses enzyme activity.
• Understanding NUDT9's structure-function relationship can illuminate TRPM2 channel mechanisms.

Purpose of the Study:

• To elucidate the structural basis of NUDT9's enzymatic activity and substrate binding.
• To investigate the structural and functional implications of mutations mimicking the TRPM2 Nudix domain.
• To compare NUDT9's catalytic mechanism with its bacterial homologues.

Main Methods:

• Determination of crystal structures of NUDT9 in the presence and absence of ribose 5'-phosphate.
• Construction and activity analysis of NUDT9 mutants, including a double point mutant (RIL) and domain-deleted constructs.
• Modeling of the NUDT9 substrate complex based on structural data and homology.

Main Results:

• NUDT9 possesses a novel N-terminal domain and a catalytic C-terminal Nudix domain, functioning as a monomer.
• Substrate binding occurs in a cleft between the NUDT9 domains, differing from its bacterial homologue.
• The RIL mutant structure explains the reduced activity of the TRPM2 ion channel domain.

Conclusions:

• NUDT9's unique domain organization and monomeric activity distinguish it from other Nudix hydrolases.
• Divergent substrate binding and active site properties suggest distinct reaction pathways between NUDT9 and E. coli ADPRase.
• Structural insights into NUDT9 provide a basis for understanding the enzymatic function of the TRPM2 channel.