In silico, structural and functional characterization of Asp-175 and Phe-169 to regulate catalytic efficiency of NAD...

NAD synthetase in methicillin resistant Staphylococcus aureus is crucial for synthesising NAD⁺ by converting deamido-NAD⁺ using ammonia and ATP. Ammonia as one of the substrates is transported as ammonium ion (NH₄⁺) through a tunnel to the enzyme active site. Tunnel forming residues are highly conserved. Using steered molecular dynamics studies, we have previously highlighted the crucial interactions of NH₄⁺ with tunnel lining residues. It has been observed that highly conserved D175 and F169 residues at active site play critical roles in the maintaining the catalytic efficiency of the enzyme. In the present work, we have assessed functional aspects of these residues using MD simulation studies, site directed mutagenesis and enzyme kinetics studies. Kinetics data suggest slow catalytic efficiency of mutants D175A and F169A with NH₄⁺ and subsequent NAD⁺ formation. Results indicate the important role of D175 in deprotonating NH₄⁺ to NH₃ which is crucial for its reaction with deamido-NAD⁺ while F169 acts as a gate to regulate NH₄⁺ movement for optimal enzyme activity.