Synthesizing and Salvaging NAD+: Lessons Learned from Chlamydomonas reinhardtii

The essential coenzyme nicotinamide adenine dinucleotide (NAD + ) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD + . Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD + is synthesized from aspartate ( de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD + synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nic otinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA ( nic2-1 and nic13-1 ) or plasmids with cloned genes ( nic1-1 and nic15-1 ) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT ( npt1-1 ) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD + . A homolog of the human SIRT6 -like gene, SRT2 , is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD + metabolism and cell longevity.