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 nicotinamide-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.
Lin
Huawen
H
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, United States of America.
Kwan
Alan L
AL
Dutcher
Susan K
SK
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D005075:Biological Evolution; D053898:Biosynthetic Pathways / Q000187:drug effects / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics; D008322:Mammals; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000096:biosynthesis*; D009536:Niacinamide / Q000494:pharmacology; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000378:metabolism; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D010940:Plant Proteins; D011725:Pyridines; C010835:3-acetylpyridine; D009243:NAD; D009536:Niacinamide; D001224:Aspartic Acid; D009612:Nicotinamide-Nucleotide Adenylyltransferase