NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae

Haemophilus influenzae has an absolute requirement for NAD (factor V) becau se it lacks almost all the biosynthetic enzymes necessary for the de novo s ynthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotin amide riboside (NR) in vitro, but little is known about the source or the m echanism of uptake of these substrates in vivo. As shown by us earlier, at feast two gene products are involved in the uptake of NAD, the outer membra ne lipoprotein e (P4), which has phosphatase activity and is encoded by hel , and a periplasmic NAD nucleotidase, encoded by nadN, It has also been obs erved that the latter gene product is essential for H, influenzae growth on media supplemented with NAD, In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilizatio n pathway, Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5 ' -nucleotidase activity. The e (P4) protein is also shown to have NMN 5 ' -nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or de letion and site-directed mutants of hel had attenuated growth and a reduced uptake pheuotype when NMN served as substrate. A hel and nadN double mutan t was only able to grow in the presence of NR whereas no uptake of NMN was observed.