Structure and function of the bacterial decapping enzyme NudC.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• RNA capping and decapping are traditionally considered eukaryotic processes.
• The redox cofactor NAD has been identified attached to bacterial small regulatory RNAs in a cap-like manner.
• Nudix hydrolase NudC functions as a NAD-decapping enzyme in vitro and in vivo.

Purpose of the Study:

• To elucidate the structural basis of NAD-decapping by bacterial Nudix hydrolase NudC.
• To understand the molecular recognition of substrate (NAD-linked RNA) and product (NMN) by NudC.
• To identify the catalytic center and quaternary structure requirements for NudC activity.

Main Methods:

• X-ray crystallography of Escherichia coli NudC in complex with NAD and NMN.
• Biochemical analysis, including site-directed mutagenesis of conserved residues.
• Enzyme activity assays to determine substrate specificity and binding characteristics.

Main Results:

• Crystal structures reveal catalytic residues and substrate/product binding modes within NudC.
• The conserved Nudix motif forms the catalytic center, requiring a homodimeric structure.
• NudC exhibits single-strand specificity, a purine preference for the 5'-terminal nucleotide, and prefers NAD-linked RNA over free NAD.
• The enzyme binds cellular RNAs non-specifically.

Conclusions:

• NudC functions as a dimeric enzyme, with its catalytic pocket formed by residues from both monomers.
• Structural and biochemical data provide a mechanistic understanding of bacterial NAD-RNA decapping.
• NudC's specificity for NAD-linked RNA suggests a role in regulating small regulatory RNAs in bacteria.