Structural and Functional Characterization of NadR from Lactococcus lactis

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• NadR is a bifunctional enzyme crucial for synthesizing nicotinamide adenine dinucleotide (NAD).
• Previous structural data from *Haemophilus influenzae* did not fully elucidate NadR's catalytic mechanism.
• Understanding NadR's mechanism is key to NAD biosynthesis pathways.

Purpose of the Study:

• To characterize the bifunctional enzyme NadR from *Lactococcus lactis*.
• To investigate the kinetic properties and structural basis of NadR's catalytic mechanism.
• To elucidate the role of ATP in the dual enzymatic activities of NadR.

Main Methods:

• Purification of NadR enzyme from *Lactococcus lactis*.
• Development of an assay to measure combined enzymatic activities.
• Kinetic analysis of substrate dependence (NR, NMN, ATP).
• X-ray crystallography of NadR from *L. lactis* (NadR_Ll) in complex with substrates and analogs.

Main Results:

• NadR exhibited hyperbolic dependence on NR and sigmoidal dependence on ATP for NR to NAD conversion.
• NMN to NAD conversion showed Michaelis-Menten kinetics for NMN but sigmoidal dependence on ATP.
• Crystal structure of NadR_Ll revealed an adenine nucleotide binding site between domains, potentially involved in regulation or substrate channeling.

Conclusions:

• The sigmoidal ATP dependence suggests cooperativity in NadR's bifunctional activity, possibly due to ATP's role in both catalytic steps.
• The identified nucleotide-binding site offers insights into potential regulatory mechanisms or substrate channeling within NadR.
• Structural and kinetic data provide a foundation for understanding NadR's role in NAD metabolism.