Structural basis of substrate recognition in human nicotinamide N-methyltransferase

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Nicotinamide N-methyltransferase (NNMT) is crucial in metabolic regulation and highly expressed in various cancers, making it a potential therapeutic target.
• Understanding NNMT's structure and function is key to developing targeted cancer therapies.

Purpose of the Study:

• To determine the crystal structure of human NNMT in a ternary complex with S-adenosyl-l-homocysteine and nicotinamide.
• To elucidate the structural basis of nicotinamide binding and identify key active site residues involved in NNMT catalysis.
• To functionally validate the role of identified active site residues through mutagenesis and enzyme kinetics.

Main Methods:

• X-ray crystallography to obtain the ternary complex structure of human NNMT at 2.7 Å resolution.
• Site-directed mutagenesis to probe the functional importance of active site residues.
• Enzyme kinetics assays to quantify the impact of mutations on NNMT activity.
• Molecular Dynamics (MD) simulations to investigate conformational effects.

Main Results:

• The crystal structure revealed the binding of S-adenosyl-l-homocysteine and nicotinamide, highlighting key active site residues.
• Mutagenesis studies showed that D197 and Y20 substitutions significantly decreased NNMT activity, while S201 and S213 had no effect.
• Enzyme kinetics confirmed the functional importance of D197 and Y20, with mutations causing 2-3 orders of magnitude decrease in k(cat)/K(m) and increased K(m) for substrates.
• MD simulations provided insights into long-range conformational effects influencing substrate binding, particularly for the D197A mutant.

Conclusions:

• The study provides a detailed structural understanding of nicotinamide binding to NNMT.
• Key active site residues, D197 and Y20, are critical for NNMT catalytic activity and substrate binding.
• These findings offer valuable insights for the rational design of NNMT inhibitors for cancer therapy.