| 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 Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis) or nicotinamide (salvage synthesis). The de novo pathway requires five different enzymes. 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 re-introducing individually cloned genes into the nic1, nic2, nic13, and nic15 mutants. NIC15 encodes L-aspartate oxidase (ASO) and carries a missense mutation (S459F). NIC2 encodes quinolate phosphoribosyltransferase (QPT) and carries a single base deletion at nucleotide 559 that generates a premature stop codon. NIC1 encodes nicotinate/ nicotinamide mononucleotide adenylyltransferase (NMNAT) and has two nucleotide changes at position 1406-1407 that results in a premature stop codon (Q346stop). NIC13 encodes NAD synthetase (NS) and carries a missense mutation (S740I). We also identified a missense mutation (L351P) in the NIC7 gene, which encodes quinolinate synthetase (QS), in the nic7 mutant that was previously characterized by Ferris (1995). Two enzymes, namely nicotinamide phosphoribosyltransferase (NAMPT) and NMNAT, are necessary components of the nicotinamide dependent salvage pathway. NMNAT is also an enzyme involved in the de novo pathway. A null NAMPT mutant has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with both the null nampt mutation and any of the nic mutations are inviable. Therefore, Chlamydomonas uses the salvage pathway found in the animal lineage and needs only one of these two pathways to synthesize NAD+. |