Structural and Functional Characterization of NadR from Lactococcus lactis

Abstract

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a ‘sink’ for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains.

Design of an in vitro multienzyme cascade system for the biosynthesis of nicotinamide mononucleotide

“…In the NRP pathway, NADR was reported to be a bifunctional enzyme, exhibiting both ribosylnicotinamide kinase activity and NMN adenylyltransferase activity. [21][22][23] For its ribosylnicotinamide kinase activity, NADR converts NR to generate NMN. [21][22][23] However, its NMN adenylyltransferase activity could further catalyze NMN to NAD + , [21][22][23] reducing the accumulation of NMN, which might be the reason why NMN production was lower using the NRP pathway.…”

“…[21][22][23] For its ribosylnicotinamide kinase activity, NADR converts NR to generate NMN. [21][22][23] However, its NMN adenylyltransferase activity could further catalyze NMN to NAD + , [21][22][23] reducing the accumulation of NMN, which might be the reason why NMN production was lower using the NRP pathway. For the APP pathway, although APT could convert AMP to generate PRPP in one step, the NMN production using the APP pathway was still much lower than that using the APH pathway under equal amounts of enzymes.…”

Enhancing the biosynthesis of nicotinamide mononucleotide in Lactococcus lactis by heterologous expression of FtnadE*

“…NadR of L. lactis NZ9000, is a bifunctional enzyme with NMN adenylytransferase and ribosylnicotinamide kinase activities using conserved domains analysis 19,20 . NadR plays an important role in the biosynthesis of NAD + , which converts nicotinamide riboside to NMN and then to NAD + 21 (Fig. 1).…”