How is NAD+ made?

How is NAD+ made in cells?

Cells make NAD+ by three pathways: the de novo pathway from tryptophan, the Preiss-Handler pathway from nicotinic acid, and the salvage pathway from nicotinamide, NMN and nicotinamide riboside. Covarrubias et al. say three independent routes keep NAD+ levels steady (2021). Reactome sorts the same reactions into the same three groups (Nicotinate metabolism, R-HSA-196807). A pathway is a chain of reactions. An enzyme is a protein that speeds up one reaction in the chain.

The routes share steps. The de novo and Preiss-Handler routes both pass through nicotinic acid mononucleotide (NAMN). The salvage route passes through nicotinamide mononucleotide (NMN). NMNAT enzymes act in all three routes (Covarrubias et al., 2021).

Cells do not only make NAD+. Covarrubias et al. describe NAD+ as made, used up and recycled all the time. Sirtuins, PARPs and CD38 use up NAD+ and leave nicotinamide behind. The salvage pathway turns that nicotinamide back into NAD+ (2021). Which enzymes use NAD+ and which make it is set out on the substrate or product page.

How does the de novo pathway make NAD+ from tryptophan?

The de novo pathway turns the amino acid tryptophan into NAD+ through the kynurenine pathway. IDO or TDO first turn tryptophan into N-formylkynurenine. A formamidase turns that into kynurenine. KMO makes 3-hydroxykynurenine, and kynureninase (KYNU) makes 3-hydroxyanthranilic acid. The enzyme 3HAO then makes a molecule called ACMS, short for α-amino-β-carboxymuconate ε-semialdehyde (Covarrubias et al., 2021; KEGG module M00912).

ACMS can fold up by itself into quinolinic acid, with no enzyme needed. The enzyme QPRT turns quinolinic acid into NAMN. NMNAT turns NAMN into NAAD, and NAD synthetase (NADSYN1) turns NAAD into NAD+. Reactome lists these last steps as QPRT, NMNAT2 and NADSYN1 (Covarrubias et al., 2021; Reactome R-HSA-196807).

KEGG module M00912 gives the same chain with enzyme numbers. It lists tryptophan 2,3-dioxygenase (EC 1.13.11.11), indoleamine 2,3-dioxygenase (EC 1.13.11.52), kynurenine formamidase (EC 3.5.1.9), kynurenine 3-monooxygenase (EC 1.14.13.9), kynureninase (EC 3.7.1.3), 3-hydroxyanthranilate 3,4-dioxygenase (EC 1.13.11.6), QPRT (EC 2.4.2.19), nicotinate-nucleotide adenylyltransferase (EC 2.7.7.18) and NAD+ synthase (EC 6.3.5.1). KEGG marks the module as complete in Homo sapiens.

Side branches of the same pathway also make kynurenic acid and picolinic acid. Serotonin is made from tryptophan by a separate route. Outside the liver, most cells lack the full set of enzymes for this route (Covarrubias et al., 2021).

What is the Preiss-Handler pathway?

The Preiss-Handler pathway makes NAD+ from nicotinic acid in three steps. The enzyme NAPRT turns nicotinic acid into NAMN. NMNAT enzymes (NMNAT1, NMNAT2 and NMNAT3) turn NAMN into NAAD. NAD synthetase (NADS) then turns NAAD into NAD+ (Covarrubias et al., 2021).

Reactome gives the same route. Nicotinic acid, also called niacin, enters the cell and becomes nicotinate D-ribonucleotide (NAMN). That molecule can then join the de novo route and become NAD+ (Reactome R-HSA-196807).

KEGG lists the NAPRT reaction (EC 6.3.4.21) as nicotinate plus 5-phosphoribosyl diphosphate plus ATP plus water, giving nicotinate D-ribonucleotide, diphosphate, ADP and phosphate.

The pathway carries the names of Jack Preiss and Philip Handler. They published a paper on the making of NAD+ in 1958. NAD+ was then called diphosphopyridine nucleotide (Preiss and Handler, 1958). Nicotinic acid and nicotinamide are both forms of vitamin B3 (Bogan and Brenner, 2008).

What is the NAD+ salvage pathway?

The salvage pathway recycles nicotinamide (NAM) back into NAD+. NAM is left over when enzymes such as sirtuins, PARPs and CD38 use up NAD+. The enzyme NAMPT turns NAM into NMN. NMNAT enzymes then turn NMN into NAD+ (Covarrubias et al., 2021).

Covarrubias et al. call NAM recycling a basic step in restoring NAD+ after it is used up (2021). Reactome names NAMPT as a central enzyme in how cells use NAM (R-HSA-196807).

KEGG lists NAMPT as EC 2.4.2.12. Its entry links nicotinamide, 5-phosphoribosyl diphosphate and nicotinamide D-ribonucleotide (NMN). KEGG lists NMNAT as EC 2.7.7.1, with the reaction ATP plus nicotinamide ribonucleotide giving diphosphate plus NAD+.

NAM does not always return to NAD+. The enzyme NNMT can add a methyl group to NAM, and the product leaves the body in urine (Covarrubias et al., 2021).

How do NMN and nicotinamide riboside become NAD+?

Both molecules join the salvage pathway and end up as NAD+ through NMNAT enzymes. Inside the cell, nicotinamide riboside (NR) becomes NMN through nicotinamide riboside kinases 1 and 2 (NRK1 and NRK2). NMNAT1, NMNAT2 and NMNAT3 then turn NMN into NAD+ (Covarrubias et al., 2021).

Outside the cell, the enzyme CD73 removes a phosphate group from NMN and makes NR. NR then enters the cell and NRK1 turns it back into NMN. Covarrubias et al. also describe a specific NMN transporter, Slc12a8, in the mouse small intestine. They say more work is needed to learn how much it matters (2021).

KEGG lists the NRK reaction (EC 2.7.1.22) as ATP plus nicotinamide riboside giving ADP plus NMN. The NAD+ vs NMN page compares the two molecules.

Where in the cell is NAD+ made?

Cells make NAD+ in more than one place, because the NMNAT enzyme comes in three forms. NMNAT1 works in the nucleus, and NMNAT2 is the form found in the cytosol, the fluid inside the cell (Covarrubias et al., 2021). Covarrubias et al. say the enzymes that make and break NAD+ are highly compartmentalised, so each cell part keeps its own pool.

NMNAT acts in all three routes. In the salvage pathway, it turns NMN into NAD+. In the de novo and Preiss-Handler pathways, it turns NAMN into NAAD, and NAD synthetase finishes the job (Covarrubias et al., 2021).

Do all living things make NAD+ the same way?

No. Some living things start from a different molecule. KEGG module M00115 starts from the amino acid aspartate and passes through quinolinate. KEGG marks it as complete in plants such as Arabidopsis thaliana. KEGG module M00912, from tryptophan, is marked as complete in Homo sapiens.

Both modules share the last steps. Quinolinate becomes nicotinate D-ribonucleotide through QPRT (EC 2.4.2.19), then nicotinate-nucleotide adenylyltransferase (EC 2.7.7.18) and NAD+ synthase (EC 6.3.1.5 or EC 6.3.5.1) finish the job. KEGG module M00115 and module M00912 list the full steps.